Therapeutic and Diagnostic Methods for Manipulating Phagocytosis Through Calreticulin and Low Density Lipoprotein-Related Receptor

ABSTRACT

Therapeutic and diagnostic methods are provided, which methods relate to the expression of calreticulin on cancer cells and hematopoietic cells.

GOVERNMENT RIGHTS

This invention was made with Government support under contracts HL058770and CA139490 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

BACKGROUND

The reticuloendothelial system (RES) is a part of the immune system. TheRES consists of the phagocytic cells located in reticular connectivetissue, primarily monocytes and macrophages. The RES consists of 1)circulating monocytes; 2) resident macrophages in the liver, spleen,lymph nodes, thymus, submucosal tissues of the respiratory andalimentary tracts, bone marrow, and connective tissues; and 3)macrophage-like cells including dendritic cells in lymph nodes,Langerhans cells in skin, and microglial cells in the central nervoussystem. These cells accumulate in lymph nodes and the spleen. The RESfunctions to clear pathogens, particulate matter in circulation, andaged or damaged hematopoietic cells.

To eliminate foreign cells or particles in the innate immune response,macrophage-mediated phagocytosis is induced when the phosphatidylserinereceptor (PSR) reacts to phosphatidylserine (PS), which can beexternalized from the membranes of dead cells, such as apoptotic andnecrotic cells. In turn, the interaction between PS and PSR plays acrucial role in the clearance of apoptotic cells by macrophages. Oncephagocytosis has been performed by macrophages, the inflammatoryresponse is downregulated by an increase in factors such as IL-10,TGF-β, and prostaglandin E2 (PGE2). The strict balance between theinflammatory and anti-inflammatory responses in both innate and adaptiveimmunity plays a critical role in maintaining cellular homeostasis andprotecting a host from extrinsic invasion.

The causal relationship between inflammation and the neoplasticprogression is a concept widely accepted. Data now support the conceptof cancer immunosurveillance—that one of the physiologic functions ofthe immune system is to recognize and destroy transformed cells.However, some tumor cells are capable of evading recognition anddestruction by the immune system. Once tumor cells have escaped, theimmune system may participate in their growth, for example by promotingthe vascularization of tumors.

Both adaptive and innate immune cells participate in the surveillanceand the elimination of tumor cells, but monocytes/macrophages may be thefirst line of defense in tumors, as they colonize rapidly and secretecytokines that attract and activate dendritic cells (DC) and naturalkiller (NK) cells, which in turn can initiate the adaptive immuneresponse against transformed cells.

Malignant cellular transformation occurs through a progression ofgenetic mutations and epigenetic reprogramming that activate oncogenesand inactivate tumor suppressor pathways leading to inheritance ofseveral hallmarks shared by most cancer cells including:self-sufficiency in growth signals, insensitivity to anti-growthsignals, tissue invasion and metastasis, poorly regulated replicativepotential, sustained angiogenesis, and evasion of cell death by avariety of pathways, including apoptosis. In addition to these cellintrinsic properties, recent evidence suggests that many cancers arealso able to evade the immune system through several distinctmechanisms.

Recently it was shown that evasion of phagocytosis through upregulationof the anti-phagocytic signal CD47 is another mechanism by which tumorcells escape immunosurveillance. CD47 is a pentaspanin cell surfaceprotein that serves as a signal inhibiting phagocytosis through ligationof its receptor SIRPa on phagocytic cells. Disruption of the CD47-SIRPαinteraction can be therapeutically targeted with a monoclonal blockingantibody against CD47, which enables phagocytosis of acute myeloidleukemia (AML), bladder cancer, and non-Hodgkin lymphoma (NHL) cells invitro and in vivo. In contrast, administration of anti-mouse CD47antibody caused minimal toxicity, despite broad expression of CD47 onnormal tissues.

CD47 has also been implicated in the regulation of phagocytosis ofapoptotic cells, as these cells become phagocytosed due to loss of CD47expression and coordinated upregulation of cell surface calreticulin.During apoptosis, cell surface calreticulin serves as a pro-phagocyticsignal by binding to its macrophage receptor, low densitylipoprotein-related protein (LRP), which leads to engulfment of thetarget cell.

Exploration of mechanisms by which cells avoid being cleared byphagocytosis can provide insight into ways for improving transplantationsuccess of hematopoietic and progenitor stem cells, and improved methodsof removing cancer cells from the body. The present invention satisfiesthese, and other, needs.

SUMMARY OF THE INVENTION

Therapeutic and diagnostic methods are provided, which methods relate tothe expression of calreticulin.

In some embodiments of the invention, the expression of calreticulin(CTR) on cancer cells, including without limitation cancer cells priorto treatment with a chemotherapeutic drug, is utilized to enhancekilling of the cancer cells. Cancer cells can be contacted with anagonist of CTR, e.g. an agonistic antibody, particularly one thatactivates LRP, in the presence of phagocytic cells in order to enhancephagocytosis of the cancer cells. In some such embodiments, the CTRagonist is administered in combination with an agent that blocks CD47signaling, e.g. soluble SIRPα, anti-CD47, and the like. Included in suchagents are bi-specific antibodies targeted to both CD47 and CTR, or CD47and LRP. Also included are agents comprising a CD47 blocking moiety andan active portion of CTR protein.

In related embodiments, cancer cells, including without limitationcancer cells prior to treatment with a chemotherapeutic drug, arecontacted with an agent that selectively binds to CRT, includingantibodies, soluble LRP, etc., which agent is optionally conjugated to atoxic moiety, e.g. a radionuclide, toxin, etc. to induce killing of thecell to which the agent has bound.

In other therapeutic methods, hematopoietic cells, including withoutlimitation HSC, hematopoietic progenitors, normal bone marrow, ormobilized peripheral blood for patients with a clinical indication forhematopoietic transplantation, are protected from phagocytosis incirculation by providing a host animal with an agent that blocks theinteraction between CRT and LRP, e.g. an antibody selective for CRT, anantibody selective for LRP, soluble CRT or LRP, a CRT blocking peptide,and the like, is administered, which blocks the pro-phagocytic signaland decreases the clearance of the hematopoietic cells from circulation.In some embodiments of the invention, the agent, e.g. peptide, solubleCRT, etc. is provided as a fusion protein, for example fused to an Fcfragment, e.g., IgG1 Fc, IgG2 Fc, Ig A Fc etc.

In another embodiment, methods are provided for targeting or depletingcancer stem cells, the method comprising contacting a population ofcells, e.g. blood from a cancer patient, with a reagent thatspecifically binds CTR in order to target or deplete the cancer stemcells. In certain aspects, the reagent is an antibody conjugated to acytotoxic agent, e.g. radioactive isotope, chemotherapeutic agent,toxin, etc. In some embodiments, the depletion is performed on an exvivo population of cells, e.g. the purging of autologous stem cellproducts (mobilized peripheral blood or bone marrow) for use inautologous transplantation for cancer patients.

Detection of calreticulin expression, e.g. cell surface protein, mRNA,etc., particularly cell surface protein, is used alone or in conjunctionwith CD47 expression for clinical diagnostic applications includingprimary diagnosis of cancers, monitoring of interval diseaseprogression, and monitoring of minimal residual disease status,including without limitation hematopoietic malignancies including acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), acutelymphocytic leukemia (ALL); and non-Hodgkin lymphoma (NHL). Cancer stemcells of interest also include carcinomas, e.g. squamous cell carcinoma,ovarian carcinoma, etc.; glioblastomas, and the like. Of interest isincluded the detection and treatment of cells prior to chemotherapeuticor radiation treatment. Detection of calreticulin on normal cells ortissues, especially hematopoietic stem and progenitor cells, in variousstates of malignancy, inflammation, and chemotherapy can guide thetiming of anti-CD47 therapies for a variety of cancers; treatments wouldbe held back during high frequency hematopoietic cell or normal tissueexpression of calreticulin until calreticulin expression levelsdecreased to minimal levels.

In a related embodiment, an agent that selectively binds to CRT, e.g.soluble LRP, anti-CTR antibody, etc. is labeled with a detectablemoiety, e.g. a fluorophore, imaging radioisotype, etc. for clinicaldiagnostic imaging applications including primary diagnosis of cancers,monitoring of interval disease progression, and monitoring of minimalresidual disease status. Imaging may be performed in vivo or ex vivo.

Detection of CTR expression is also used in prognosis of cancer, whereincreased levels of CTR are shown to be associated with a worse clinicalprognosis in multiple human malignancies.

Of particular interest is the detection of CRT expression on cancer stemcells, where it has been found that CRT expression segregates withtumorigenicity. Expression of CRT is used alone or in combination withother cancer stem cell markers, e.g. CD47, CD44, etc. to identify,target and/or isolated cancer stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Cell surface calreticulin is expressed on cancer, but notnormal, stem and progenitor cells (A) Cell surface calreticulinexpression was determined by flow cytometry on primary human patientsamples from several hematologic cancer types and normal cellcounterparts including normal bone marrow (NBM, n=9), normal peripheralblood (NPB, n=3), acute myeloid leukemia (AML, n=8), acute lymphoblasticleukemia (ALL, n=21), chronic myeloid leukemia (CML, n=13), andnon-Hodgkin lymphoma (NHL, n=7). (B) A similar analysis as in A wasperformed for solid tumors (glioblastoma, n=9; transitional cell bladdercarcinoma, n=8; serous papillary ovarian carcinoma, n=9) and normalhuman fetal tissues (neurons, n=3; astrocytes, n=6, bladder cells, n=6).ESA+ urothelium was analyzed for normal fetal bladder. Primary humanbladder cancer patient samples and samples that had been passaged oncein mice were used for profiling. (C and D) Cell surface calreticulinexpression was determined on normal stem and progenitor cells,lymphocytes, and cancer stem and progenitor cells. Each symbolrepresents a different patient sample. Patient samples tested: NBM=10,AML=8, CML=13, bladder cancer=8, glioblastoma=8. NBMHSC=CD34+CD38-CD90+Lin−, AML LSC=CD34+CD38-CD90-Lin−,GMP=CD34+CD38+IL3rα+CD45RA+, CMP=CD34+CD38+IL3rα+CD45RA−. (E)Calreticulin expression did not differ between bulk and cancer stem cellpopulations for either bladder cancer (p=0.54) or glioblastoma (p=0.14).Bladder cancer CSC=CD44+Lin− (8), glioblastoma CSC=CD133+Lin− (22, 23).Annexin V-positive cells were excluded in the analysis of all samples.

FIG. 2: Increased CD47 Expression on Cancer Cells Protects Them fromCalreticulin-Mediated Phagocytosis (A) Correlation between cell surfacecalreticulin and CD47 expression was determined for human cancer celllines (top left) and primary human normal and cancer samples (top right,bottom panels). Expression was calculated as mean fluorescence intensitynormalized over isotype control and for cell size. Pearson correlation(r) and p-value is shown for each correlation. Top left panel: bluesolid circle=HL60, blue open circle=Kasumil, blue open invertedtriangle=MOLM13, blue open diamond=KG-1, red triangle=Jurkat, red solidsquare=CCRF-CEM, red open square=CCRF-HSB2, red diamond=MOLT4, blackstar=Raji, black open diamond=SUDHL6, black open triangle=Daudi, blackx=U937, green plus=639V, green open diamond=HT1197, green invertedtriangle=UMUC3. (B) CD47 protein expression was determined by flowcytometry on Raji cells transduced with lentiviruses encoding shRNACD47-knockdown constructs (shCD47) or controls. (C) Relative CD47expression levels were quantified by comparing MFI to wild type Rajicells. (D) Raji cell clones were incubated with human macrophages inmedia alone or with CRT blocking peptide for 2 hours, after whichphagocytosis was analyzed by fluorescence microscopy. Knockdown of CD47in Raji cells (shCD47-1,-2) resulted in increased phagocytosis comparedto untransduced Raji cells. No difference in phagocytosis was observedbetween untransduced and GAPD control-transduced Raji cells (p=0.45)Blockade of calreticulin on CD47-knockdown Raji cells completelyabrogated phagocytosis. (E) MOLM-13 cells, a CD47-deficient human AMLcell line, were incubated with human macrophages for two hours with theindicated peptides and monitored for phagocytosis as above. Significantphagocytosis was observed with IgG1 isotype control, while blockade ofcalreticulin or LRP reduced levels of phagocytosis (p=0.03 and p=0.01,respectively). Conditions were performed in triplicate; data presentedas mean±SD. *p<0.05, **p<0.005, ***p<0.0005 (2-tailed Student's t-test).

FIG. 3: Cell Surface Calreticulin is the Dominant Pro-Phagocytic Signalon Several Human Cancers and is Required for Anti-CD47 Antibody-MediatedPhagocytosis (A) Primary human AML cells were fluorescently-labeled withCFSE and incubated with human macrophages in the presence of theindicated antibodies/peptides for 2 hours, after which phagocytosis wasanalyzed by fluorescence microscopy. Arrows indicate phagocytosis. (B)Cells from several normal human tissue types were incubated with humanmacrophages in the presence of the indicated antibodies and monitoredfor phagocytosis. No difference in phagocytosis was detected betweenIgG1 isotype control and anti-CD47 antibody incubation (p=0.77). (C)Primary human cancer cells were incubated with human macrophages in thepresence of the indicated antibodies/peptides for two hours andmonitored for phagocytosis. Each data point represents a differentpatient sample. Compared to IgG1 isotype control, incubation withanti-CD47 antibody enabled phagocytosis of cancer cells (p<0.0001) whileincubation with calreticulin blocking peptide (p=0.37) or RAP, an LRPinhibitor (p=0.67), did not enable phagocytosis. In the presence ofanti-CD47 antibody, incubation of cancer cells with either calreticulinblocking peptide or RAP completely abrogated anti-CD47 antibody-mediatedphagocytosis (p=0.77 and p=0.16, respectively compared to IgG1 isotypecontrol). *****p<0.00001 (2-sided Student's t-test). (D) A positivecorrelation was observed between cell surface CRT expression and degreeof anti-CD47 antibody mediated phagocytosis (Pearson's correlationcoefficient is shown). Each point represents a distinct patient samplethat was incubated in the same in vitro phagocytosis assay. (E) HumanNBM cells were incubated with human macrophages in the presence of theindicated antibodies or protein. Exogenous CRT enabled increasedphagocytosis of NBM cells compared to vehicle control (p=0.05). Nodifference in phagocytosis was observed between IgG1 isotype control andanti-CD47 antibody (p=0.49). Conditions were performed in triplicate;data presented as mean±SD.

FIG. 4: Increased calreticulin expression confers a worse clinicalprognosis in multiple human malignancies. Stratification of clinicaloutcomes based on the level of expression of calreticulin mRNA is shownin previously described cohorts of patients with diverse malignanciesincluding neuroblastoma (A,B), superficial or invasive bladder cancer(C,D), and mantle cell lymphoma (E,F). Patients were divided intocalreticulin high and low expression groups based on median calreticulinexpression with Kaplan-Meier analyses of patient outcome shown. Hazardratios (HR) and log-rank p values are shown for the relationship ofoutcomes to continuous expression of calreticulin using a univariate Coxregression model. HR, 95% confidence intervals, and log-rank p valuesfor calreticulin expression as a dichotomous variable are shown intable 1. Description of clinical datasets is shown in table 1.

FIG. 5: Live calreticulin positive cancer cells form tumors in vivo (A)CRT− and CRT+ LSC from human AML patient samples were sorted to 100%purity by FACS. (B) 5,000 CRT− or CRT+ AML LSC were transplanted intothe facial vein of sublethally-irradiated newborn NSG. Eight weeks latermice were sacrificed and analyzed for AML bone marrow engraftment. EqualAML engraftment (as shown by human CD45+ chimerism) was observed in bothCRT− and CRT+ AML LSC. Representative data are shown. (C) CRT− and CRT+primary human bladder cancer cells from mouse xenografts were sorted byFACS to >99% purity. (D,E) 10,000 CRT− or CRT+ bladder cancer cells weretransplanted subcutaneously onto the flanks of NSG mice. Eight weekslater solid tumor growth was equal in mice transplanted with CRT- andCRT+ cells (D, top panels). CRT+ and CRT-tumors were excised (D, bottompanels), with no difference in tumor weight (E, p=0.63).

FIG. 6: Cell surface CRT correlates with ERp57 expression on tumorcells. (A) A positive correlation between cell surface CRT and ERp57expression was found on primary human tumors or cancer cell lines usingPearson's correlation coefficient. (B) These cells were analyzed forcell surface expression on ERp57, CRT, and CD47 by flow cytometry. Arepresentative staining profile is shown for Raji cells. A greaterpercentage of CRT positive cells expressed ERp57 (third panel) comparedto CRT negative cells (second panel). CRT+ERp57+ cells also expressedCD47 (fourth panel) in similar levels to the bulk cell population (datanot shown). (C) Cell surface ERp57 expression was quantified in CRT+ andCRT− cell populations from several tumor types. A greater percentage ofCRT+ cells expressed ERp57 compared to CRT− counterparts (p=0.0006).Each symbol represents a distinct tumor sample. Samples shown were frompatient samples or the following cell lines: blue square=Jurkat, greeninverted triangle=Raji, green diamond=SUDHL4. All cells profiled in(A-C) were excluded for annexin V-positive cells.

FIG. 7: Live calreticulin positive cells from normal human tissues havehigher levels of CD47 compared to calreticulin negative cells. (A,B)Left panel: bulk normal human bone marrow cells (A) or normal humanfetal bladder (ESA positive) urothelial cells (B) were profiled for cellsurface calreticulin expression by flow cytometry. Right panel: cellsurface calreticulin-negative and -positive cells were profiled for CD47expression, demonstrating higher CD47 expression oncalreticulin-positive cells. Annexin V-positive cells were excluded fromthe analysis of both bulk normal human bone marrow cells and fetalbladder cells. Data is representative of several samples.

FIG. 8: Calreticulin expression is unaffected by CD47 shRNA knockdown inRaji cells. Raji cells were transduced with lentiviral constructsencoding shRNA directed against CD47 (Raji shCD47-1, shCD47-2) or a GAPDcontrol (Raji GAPD). Cell surface calreticulin expression was determinedby flow cytometry and demonstrated no difference on wild type,untransduced Raji cells compared to Raji cells transduced with eitherGAPD control, shCD47-1, or shCD47-2 lentivirus. Cell surfacecalreticulin expression for MOLM13 cells is also shown.

FIG. 9: CD47 is expressed on normal human cells. (A-B) CD47 expressionwas determined by flow cytometry on normal human hematopoietic cells (A)and fetal tissue cells (B) demonstrating expression on all normal cellsprofiled. Flow cytometry plots are from a representative sample of eachnormal tissue cell type.

FIG. 10: Abrogation of anti-CD47 antibody-mediated phagocytosis is dosedependent on calreticulin blockade. (A) Cell surface CRT and CD47expression was determined by flow cytometry on Jurkat cells, a T cellleukemia cell line. (B) Jurkat cells were incubated with humanmacrophages in the presence of the indicated antibodies and blockingpeptides, and phagocytosis was determined by fluorescence microscopy.Anti-CD47 antibody was used at 10 μg/ml. CRT blocking peptideconcentrations are shown as μg/ml. Each condition was performed intriplicate. Data is expressed as mean±SD.

FIG. 11: Model for the integration of pro (CRT)- and anti(CD47)-phagocytic signals on normal and tumor cells at steady state andduring anti-CD47 antibody therapy. (A,B) At steady state, tumor, normal,and damaged cells express varying levels of cell surface CD47 and CRT,and it is the integration of both signals that determines whether thetarget cell will be phagocytosed. Tumor cells express CRT, but alsohigher levels of CD47 that delivers a dominant negative phagocyticsignal (minus sign), leading to evasion of phagocytosis. In contrast,normal cells express lower levels of CD47, but do not express CRT, andthus no phagocytosis occurs. Lastly, damaged or apoptotic cells exhibithigh levels of CRT expression, and this positive phagocytic signal (plussign) dominates over low CD47 expression, leading to phagocytosis(dashed arrow). (C,D) During anti-CD47 antibody therapy, the negativephagocytic stimulus (CD47) is blocked. In tumor cells, this unmasks thepositive phagocytic signal (CRT), leading to phagocytosis. In contrast,normal cells are not phagocytosed since the positive phagocytic stimulus(CRT) is absent.

FIG. 12. CRT was knocked down in HL60 human leukemia cells by infectionwith lenti viruses expressing control shRNA (control KD) or shRNAtargeting CRT (CRT KD). (left Panel) Knockdown (KD) efficiency wasevaluated by western blot (IB) with anti-CRT antibody, and GAPDH wasused as a loading control. (Right Panel) HL60 cells treated with controlKD, CRT KD or CRT blocking peptide were treated with anti-CD47 antibody(B6H12) or IgG control, and incubated with J774 macrophages forphagocytosis as described above. The phagocytic index of the CRT KDcells was about half of the control KD cells and the CRT peptide blocked˜75% of the phagocytosis observed when treated with anti CD47 antibodyindicating that reducing or blocking the prophagocytic signal CRT allowsphagocytosis to proceed in the presence of antiCD47 antibody.

FIG. 13. CRT and CD47 expression were examined in MCF-10A (10A, normalhuman breast epithelial cell line), MCF-7 (7, tumorigenic human breastcancer cell line) and MDA-MB-231 (231, metastatic human breast cancercell line). (Lower left) Histograms of CD47 expression as determined byflow cytometry on each of the above cell lines with FITC-conjugatedanti-CD47 antibody and isotype matched control antibody indicating thattumorigenic MCF-7 cells express high levels of CD47. The metastatic(231) breast cancer line expressed lower level of CD47, but slightlyhigher than the normal breast cell line (10A). (Lower Right) Surfacebiotinylation to detect surface CRT expression, Sulfo-NHS-SS-Biotin wasused to label cell surface proteins and cell lysate was then incubatedwith neutravidin beads to collect biotin labeled proteins. Surfacebiotin-labeled CRT was detected by Western analysis with anti-CRTantibody and cytosolic protein GAPDH was used as a negative control.Consistent with the flow cytometry data, higher levels of surface CRTwere observed in tumorigenic MCF-7 cells than in the normal (10A) ormetastatic (231) breast cancer lines.

FIGS. 14A-B. Calreticulin on the cell surface of cells undergoingapoptosis bind to LRP1 protein. (A) Molm 13 cells (human acute myelomacells) treated with apoptotic agent staurosporine express calreticulin.Live cells were stained with anti-calreticulin (CRT) antibody conjugatedby a fluorophore. Histrograms represent an increase in cell surfacecalreticulin compared to isotype control, analyzed by flow cytometry.(B) Molm 13 cells treated with apoptotic agent staurosporine bind toLRP1 cluster IV-Fc protein. Live cells were stained with LRP1 clusterIV-Fc protein followed by a secondary anti-Fc antibody conjugated to afluorophore. Histrograms represent an increase in the binding of LRP1cluster IV-Fc protein. This binding can be blocked with calreticulinblocking peptide (CRT BP) and with an LRP1 inhibitor, receptorassociated protein (RAP).

FIGS. 15A-B. Calreticulin is expressed on the cell surface of viable,non-apoptotic cells tumor cells. Pancreatic cancer cell line and primarytumors express calreticulin. Paca-2 cells were fixed and stained withanti-calreticulin (CRT) and secondary antibody (A). Arrows showexpression of calreticulin on the cell surface. A human primaryneuroendocrine tumor sample was sectioned, fixed and stained withanti-CRT, and cell surface marker EpCAM, and nuclear stain Dapi (B).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Under normal physiologic conditions, cellular homeostasis is partlyregulated by balancing pro- and anti-phagocytic signals. For example,the anti-phagocytic protein CD47 is highly expressed on several humancancers including acute myeloid leukemia, non-Hodgkin lymphoma, andbladder cancer, where it allows cancer cells to evade phagocytosis bythe innate immune system. It has been found that blockade of CD47enables phagocytosis of cancer cells and leads to in vivo tumorelimination. In order for these target cells to be phagocytosed uponblockade of an anti-phagocytic signal, it is shown herein that the cellsmust also display a pro-phagocytic signal, which is identified ascalreticulin. CRT is highly expressed on the cell surface of multiplehuman cancers, including without limitation, acute myeloid andlymphoblastic leukemias, chronic myeloid leukemia, non-Hodgkin lymphoma(NHL), bladder cancer, glioblastoma, and ovarian cancer, but minimallyexpressed on most normal cells.

Increased CD47 expression was found to be correlated with highcalreticulin levels on cancer cells, and was necessary for protectionfrom calreticulin-mediated phagocytosis. Phagocytosis induced byanti-CD47 antibody required the interaction of target cell calreticulinwith its receptor low density lipoprotein-receptor related protein (LRP)on phagocytic cells, as blockade of the calreticulin/LRP interactionprevented anti-CD47 antibody mediated phagocytosis. Increasedcalreticulin expression is an adverse prognostic factor in diversetumors including neuroblastoma, bladder cancer, and NHL.

Methods are provided to manipulate phagocytosis of cells, particularlycancer cells and hematopoietic cells, by modulating CRT activity.Methods are additionally provided for detection and monitoring of cancercells by determining expression of CRT; where such cancer cells may beselectively targeted for ADCC, chemotherapy, etc. by agents thatspecifically bind CRT.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

Calreticulin.

Calreticulin is a multifunctional protein of 417 amino acids, molecularweight 48 kDa, that binds Ca²⁺ ions, rendering it inactive. The Ca²⁺ isbound with low affinity, but high capacity, and can be released on asignal. Calreticulin can be located in storage compartments associatedwith the endoplasmic reticulum, where it binds to misfolded proteins andprevents them from being exported to the Golgi apparatus. Calreticulinis also found in the nucleus, suggesting that it may have a role intranscription regulation. Calreticulin binds to the synthetic peptideKLGFFKR, which is almost identical to an amino acid sequence in theDNA-binding domain of the superfamily of nuclear receptors.

The gene symbol for calreticulin is CALR, and the human sequences may beaccessed at Pubmed as follows: Protein Accession# NP_(—)004334;Nucleotide Accession#: NM_(—)004343.

Gardai et al. (2005) stated that calreticulin on the surface ofapoptotic cells serves as a recognition and clearance ligand byactivating the internalization receptor LRP on the responding phagocytecell surface. Using mouse and human cells, it was found that the surfaceexpression of calreticulin increased and calreticulin was redistributedduring apoptosis, possibly enhancing stimulation of LRP on thephagocyte. In addition, CD47 on the apoptotic cell surface was alteredand/or lost, which reduced the activation of SIRP-alpha on thephagocytic cell surface, resulting in phagocytosis.

In CT26 mouse colon cancer cells, Obeid et al. (2007) demonstrated thatanthracyclins induced immunogenic cell death by way of a rapid,pre-apoptotic translocation of calreticulin to the cell surface.Blockade or knockdown of CALR suppressed phagocytosis ofanthracyclin-treated tumor cells by dendritic cells and abolished theirimmunogenicity in mice. Anthracyclin-induced CALR translocation wasmimicked by inhibition of the protein phosphatase-1/Gadd34 complex.Administration of recombinant CALR or inhibitors of Pp1/Gadd34 restoredimmunogenicity of cell death elicited by etoposide and mitomycin C andenhanced their antitumor effects in vivo.

Low Density Lipoprotein Receptor-Related Protein (LRP; CD91).

The low density lipoprotein receptor-related protein (LRP) is a4,544-amino acid protein containing a single transmembrane segment, witha high degree of sequence identity to the LDL receptor. GP96, HSP90,HSP70, and calreticulin use CD91 as a common receptor. The human geneticsequences may be accessed at Pubmed as follows: Nucleotide Accession#:NM_(—)002332.2 GI:126012561.

Calreticulin Binding Agents.

Agents that specifically bind to calreticulin (CRT) are of interest asdetectable markers for imaging and diagnosis, as therapeutic agents fortargeted delivery of chemotherapeutic moieties; as therapeutic agentsfor antibody dependent cytotoxicity (ADCC); as agonists for enhancingthe pro-phagocytic activity of CRT; and as inhibitors of CRT activity,e.g. by blocking the interaction of CRT and LRP. The term “specificbinding member” or “binding member” as used herein refers to a member ofa specific binding pair, i.e. two molecules, usually two differentmolecules, where one of the molecules (i.e., first specific bindingmember) through chemical or physical means specifically binds to theother molecule (i.e., second specific binding member). CRT bindingagents useful in the methods of the invention include analogs,derivatives and fragments of the original specific binding member, e.g.Fab fragments of antibodies, etc.

CRT binding agents that act as inhibitors include blocking peptides,which are commercially available and known in the art (see Urade et al.(2004) Biochemistry 43 (27), 8858-8868, herein specifically incorporatedby reference; and commercial suppliers including, inter alia, AvivaSystems Biology; MBL International Corporation), including withoutlimitation the peptides KLGFFKR; andKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPQA KDEL; blockingoligosaccharides (see Arai et al. (2005) Chembiochem 6(12):2281-2289,herein specifically incorporated by reference); and blocking antibodies(see Urade et al., supra. and peptide sources).

In a preferred embodiment, the specific binding member is an antibody.The term “antibody” or “antibody moiety” is intended to include anypolypeptide chain-containing molecular structure with a specific shapethat fits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. Antibodies utilized in the present inventionmay be polyclonal antibodies, although monoclonal antibodies arepreferred because they may be reproduced by cell culture orrecombinantly, and can be modified to reduce their antigenicity.

The phrase “bispecific antibody” refers to a synthetic or recombinantantibody that recognizes more than one protein. Examples includebispecific antibodies 2B1, 520C9×H22, mDX-H210, and MDX447. Bispecificantibodies directed against a combination of epitopes, will allow forthe targeting and/or depletion of cellular populations expressing thecombination of epitopes. Exemplary bi-specific antibodies include thosetargeting a combination of CALRETICULIN and a cancer cell marker, suchas, CD96, CD97, CD99, PTHR2, HAVCR2 etc. Generation of bi-specificantibody is described in the literature, for example, in U.S. Pat. No.5,989,830, U.S. Pat. No. 5,798,229, which are incorporated herein byreference.

Polyclonal antibodies can be raised by a standard protocol by injectinga production animal with an antigenic composition. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Freund's, Freund'scomplete, oil-in-water emulsions, etc.) When a smaller peptide isutilized, it is advantageous to conjugate the peptide with a largermolecule to make an immunostimulatory conjugate. Commonly utilizedconjugate proteins that are commercially available for such use includebovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In orderto raise antibodies to particular epitopes, peptides derived from thefull sequence may be utilized. Alternatively, in order to generateantibodies to relatively short peptide portions of the protein target, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as ovalbumin, BSA or KLH.

Alternatively, for monoclonal antibodies, hybridomas may be formed byisolating the stimulated immune cells, such as those from the spleen ofthe inoculated animal. These cells are then fused to immortalized cells,such as myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. In addition, the antibodies orantigen binding fragments may be produced by genetic engineering.Humanized, chimeric, or xenogeneic human antibodies, which produce lessof an immune response when administered to humans, are preferred for usein the present invention.

Antibodies that have a reduced propensity to induce a violent ordetrimental immune response in humans (such as anaphylactic shock), andwhich also exhibit a reduced propensity for priming an immune responsewhich would prevent repeated dosage with the antibody therapeutic orimaging agent are preferred for use in the invention. These antibodiesare preferred for all administrative routes. Thus, humanized, chimeric,or xenogenic human antibodies, which produce less of an immune responsewhen administered to humans, are preferred for use in the presentinvention.

Chimeric antibodies may be made by recombinant means by combining themurine variable light and heavy chain regions (VK and VH), obtained froma murine (or other animal-derived) hybridoma clone, with the humanconstant light and heavy chain regions, in order to produce an antibodywith predominantly human domains. The production of such chimericantibodies is well known in the art, and may be achieved by standardmeans (as described, e.g., in U.S. Pat. No. 5,624,659, incorporatedfully herein by reference). Humanized antibodies are engineered tocontain even more human-like immunoglobulin domains, and incorporateonly the complementarity-determining regions of the animal-derivedantibody. This is accomplished by carefully examining the sequence ofthe hyper-variable loops of the variable regions of the monoclonalantibody, and fitting them to the structure of the human antibodychains. Although facially complex, the process is straightforward inpractice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully hereinby reference. Alternatively, single chain antibodies (Fv, as describedbelow) can be produced from phage libraries containing human variableregions. See U.S. Pat. No. 6,174,708, incorporated fully herein byreference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ficin, pepsin, papain, or otherprotease cleavage. “Fragment” or minimal immunoglobulins may be designedutilizing recombinant immunoglobulin techniques. For instance “Fv”immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

Fv fragments are heterodimers of the variable heavy chain domain (V_(H))and the variable light chain domain (V_(L)). The heterodimers of heavyand light chain domains that occur in whole IgG, for example, areconnected by a disulfide bond. Recombinant Fvs in which V_(H) and V_(L)are connected by a peptide linker are typically stable, see, forexample, Huston et al., Proc. Natl. Acad, Sci. USA 85:5879-5883 (1988)and Bird et al., Science 242:423-426 (1988), both fully incorporatedherein, by reference. These are single chain Fvs which have been foundto retain specificity and affinity and have been shown to be useful forimaging tumors and to make recombinant immunotoxins for tumor therapy.Any of these minimal antibodies may be utilized in the presentinvention, and those which are humanized to avoid HAMA reactions arepreferred for use in embodiments of the invention.

In addition, derivatized immunoglobulins with added chemical linkers,detectable moieties, e.g. fluorescent dyes, enzymes, radioisotopes,substrates, chemiluminescent moieties, or specific binding moieties,e.g. streptavidin, avidin, biotin, etc. may be utilized in the methodsand compositions of the present invention. For convenience, the term“antibody” or “antibody moiety” will be used throughout to generallyrefer to molecules which specifically bind to an epitope of the targetedprotein, although the term will encompass all immunoglobulins,derivatives, fragments, recombinant or engineered immunoglobulins, andmodified immunoglobulins, as described above.

Candidate binding agents can be tested for activity by any suitablestandard means. As a first screen, the antibodies may be tested forbinding against the target antigen utilized to produce them. As a secondscreen, candidate agents may be tested for binding to an appropriatecell, e.g. cancer cell, hematopoietic cell, etc. For these screens, thecandidate antibody may be labeled for detection (e.g., with fluoresceinor another fluorescent moiety, or with an enzyme such as horseradishperoxidase). After selective binding to the target is established, thecandidate agent may be tested for appropriate activity (i.e., theability to decrease tumor cell growth and/or to aid in visualizing tumorcells) in an in vivo model.

The antibodies for use in the present invention may have utility withoutconjugation, e.g. when the native activity of the target protein isaltered in the tumor cell, when the antibody binding is sufficient toactivate ADCC, etc. Such antibodies, which may be selected as describedabove, may be utilized without further modification. However, theconjugation of cytotoxic or imaging agents is yet another preferredembodiment when utilizing these antibodies because the added moietiesadd functionality to the therapeutic.

Thus, in many preferred embodiments of the invention, the antibodies maybe coupled or conjugated to one or more therapeutic cytotoxic or imagingmoieties. As used herein, “cytotoxic moiety” simply means a moiety whichinhibits cell growth or promotes cell death when proximate to orabsorbed by the cell. Suitable cytotoxic moieties in this regard includeradioactive isotopes (radionuclides), chemotoxic agents such asdifferentiation inducers and small chemotoxic drugs, toxin proteins, andderivatives thereof. As utilized herein, “imaging moiety” means a moietywhich can be utilized to increase contrast between a tumor and thesurrounding healthy tissue in a visualization technique (e.g.,radiography, positron-emission tomography, magnetic resonance imaging,direct or indirect visual inspection). Thus, suitable imaging moietiesinclude radiography moieties (e.g. heavy metals and radiation emittingmoieties), positron emitting moieties, magnetic resonance contrastmoieties, and optically visible moieties (e.g., fluorescent orvisible-spectrum dyes, visible particles, etc.).

In general, therapeutic or imaging agents may be conjugated to theantibody by any suitable technique, with appropriate consideration ofthe need for pharmokinetic stability and reduced overall toxicity to thepatient. A therapeutic agent may be coupled to a suitable antibodymoiety either directly or indirectly (e.g. via a linker group). A directreaction between an agent and an antibody is possible when eachpossesses a functional group capable of reacting with the other. Forexample, a nucleophilic group, such as an amino or sulfhydryl group, maybe capable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide). Alternatively, a suitable chemicallinker group may be used. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on a moiety or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of moieties, or functional groupson moieties, which otherwise would not be possible.

Where a cytotoxic moiety is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell, or which is gradually cleavable over timein the extracellular environment. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof a cytotoxic moiety agent from these linker groups include cleavage byreduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014), byhydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No.4,638,045), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789).

Preferred radionuclides for use as cytotoxic moieties are radionuclideswhich are suitable for pharmacological administration. Suchradionuclides include ¹²³I, ¹²⁵I, ¹³¹I, ⁹⁰Y, ²¹¹At, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re,²¹²Pb, and ²¹²Bi. Preferred chemotoxic agents include small-moleculedrugs such as carboplatin, cisplatin, vincristine, taxanes such aspaclitaxel and doceltaxel, hydroxyurea, gemcitabine, vinorelbine,irinotecan, tirapazamine, matrilysin, methotrexate, pyrimidine andpurine analogs, and other suitable small toxins known in the art.Preferred chemotoxin differentiation inducers include phorbol esters andbutyric acid. Chemotoxic moieties may be directly conjugated to theantibody moiety via a chemical linker, or may encapsulated in a carrier,which is in turn coupled to the antibody moiety. Preferred toxinproteins for use as cytotoxic moieties include ricins A and B, abrin,diphtheria toxin, bryodin 1 and 2, momordin, trichokirin, cholera toxin,gelonin, Pseudomonas exotoxin, Shigella toxin, pokeweed antiviralprotein, and other toxin proteins known in the medicinal biochemistryarts.

Preferred radiographic moieties for use as imaging moieties in thepresent invention include compounds and chelates with relatively largeatoms, such as gold, iridium, technetium, barium, thallium, iodine, andtheir isotopes. It is preferred that less toxic radiographic imagingmoieties, such as iodine or iodine isotopes, be utilized in thecompositions and methods of the invention. Positron emitting moietiesfor use in the present invention include ¹⁸F, which can be easilyconjugated by a fluorination reaction with the antibody. Magneticresonance contrast moieties include chelates of chromium(III),manganese(II), iron(II), nickel(II), copper(II), praseodymium(III),neodymium(III), samarium(III) and ytterbium(III) ion. Optically visiblemoieties for use as imaging moieties include fluorescent dyes, orvisible-spectrum dyes, visible particles, and other visible labelingmoieties. Fluorescent dyes such as fluorescein, coumarin, rhodamine,bodipy Texas red, and cyanine dyes, are useful when sufficientexcitation energy can be provided to the site to be inspected visually.Endoscopic visualization procedures may be more compatible with the useof such labels. Acceptable dyes include FDA-approved food dyes andcolors, which are non-toxic, although pharmaceutically acceptable dyeswhich have been approved for internal administration are preferred.

In one embodiment of the invention, the agent, or a pharmaceuticalcomposition comprising the agent, is provided in an amount effective todetectably inhibit the binding of calreticulin to LRP present on thesurface of phagocytic cells. The effective amount is determined viaempirical testing routine in the art. The effective amount may varydepending on the number of cells being transplanted, site oftransplantation and factors specific to the transplant recipient.

Calreticulin “mimetics” and “agonists” include molecules that functionsimilarly to or potentiate CRT by binding and activating LRP receptor.Molecules useful as CRT mimetics include derivatives, variants, andbiologically active fragments of naturally occurring CRT. Moleculesuseful as agonists include antibodies and other agents that act toenhance the pro-phagocytic activity of CRT.

A “variant” polypeptide means a biologically active polypeptide asdefined below having less than 100% sequence identity with a nativesequence polypeptide. Such variants include polypeptides wherein one ormore amino acid residues are added at the N- or C-terminus of, orwithin, the native sequence; from about one to forty amino acid residuesare deleted, and optionally substituted by one or more amino acidresidues; and derivatives of the above polypeptides, wherein an aminoacid residue has been covalently modified so that the resulting producthas a non-naturally occurring amino acid. Ordinarily, a biologicallyactive variant will have an amino acid sequence having at least about90% amino acid sequence identity with a native sequence polypeptide,preferably at least about 95%, more preferably at least about 99%. Thevariant polypeptides can be naturally or non-naturally glycosylated,i.e., the polypeptide has a glycosylation pattern that differs from theglycosylation pattern found in the corresponding naturally occurringprotein. The variant polypeptides can have post-translationalmodifications not found on the natural CRT protein.

Fragments of soluble CRT, particularly biologically active fragmentsand/or fragments corresponding to functional domains, are of interest.Fragments of interest will typically be at least about 10 aa to at leastabout 15 aa in length, usually at least about 50 aa in length, but willusually not exceed about 142 aa in length, where the fragment will havea stretch of amino acids that is identical to CRT. A fragment “at least20 aa in length,” for example, is intended to include 20 or morecontiguous amino acids from, for example, the polypeptide encoded by acDNA for CRT. In this context “about” includes the particularly recitedvalue or a value larger or smaller by several (5, 4, 3, 2, or 1) aminoacids. The protein variants described herein are encoded bypolynucleotides that are within the scope of the invention. The geneticcode can be used to select the appropriate codons to construct thecorresponding variants. The polynucleotides may be used to producepolypeptides, and these polypeptides may be used to produce antibodiesby known methods.

A “fusion” polypeptide is a polypeptide comprising a polypeptide orportion (e.g., one or more domains) thereof fused or bonded toheterologous polypeptide. A fusion soluble CRT protein, for example,will share at least one biological property in common with a nativesequence soluble CRT polypeptide. Examples of fusion polypeptidesinclude immunoadhesins, as described above, which combine a portion ofthe CRT polypeptide with an immunoglobulin sequence, including animmunoglobulin specific for CD47, and epitope tagged polypeptides, whichcomprise a soluble CRT polypeptide or portion thereof fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with biological activity of the CRTpolypeptide. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 6-60 amino acid residues.

A “functional derivative” of a native sequence polypeptide is a compoundhaving a qualitative biological property in common with a nativesequence polypeptide. “Functional derivatives” include, but are notlimited to, fragments of a native sequence and derivatives of a nativesequence polypeptide and its fragments, provided that they have abiological activity in common with a corresponding native sequencepolypeptide. The term “derivative” encompasses both amino acid sequencevariants of polypeptide and covalent modifications thereof. Derivativesand fusion of soluble CRT find use as CRT mimetic molecules.

In vitro assays for calreticulin biological activity include, e.g.phagocytosis of porcine cells by human macrophages, binding to LRP, etc.A candidate agent useful as a calreticulin agonist mimetic results inthe down regulation of phagocytosis by at least about 10%, at leastabout 20%, at least about 50%, at least about 70%, at least about 80%,or up to about 90% compared to level of phagocytosis observed in absenceof candidate agent.

A plurality of assays may be run in parallel with differentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in binding.

By “manipulating phagocytosis” is meant an up-regulation or adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention. Thus in the context of decreasingphagocytosis of circulating hematopoietic cells, particularly in atransplantation context, manipulating phagocytosis means adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arethree main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

Hematopoietic stem cells (HSC), as used herein, refers to a populationof cells having the ability to self-renew, and to give rise to allhematopoietic lineages. Such cell populations have been described indetail in the art. Hematopoietic progenitor cells include the myeloidcommitted progenitors (CMP), the lymphoid committed progenitors (CLP),megakaryocyte progenitors, and multipotent progenitors. The earliestknown lymphoid-restricted cell in adult mouse bone marrow is the commonlymphocyte progenitor (CLP), and the earliest known myeloid-restrictedcell is the common myeloid progenitor (CMP). Importantly, these cellpopulations possess an extremely high level of lineage fidelity in invitro and in vivo developmental assays. A complete description of thesecell subsets may be found in Akashi et al. (2000) Nature 404(6774):193,U.S. Pat. No. 6,465,247; and published application U.S. Ser. No.09/956,279 (common myeloid progenitor); Kondo et al. (1997) Cell91(5):661-7, and International application WO99/10478 (common lymphoidprogenitor); and is reviewed by Kondo et al. (2003) Annu Rev Immunol.21:759-806, each of which is herein specifically incorporated byreference. The composition may be frozen at liquid nitrogen temperaturesand stored for long periods of time, being capable of use on thawing.For such a composition, the cells will usually be stored in a 10% DMSO,50% FCS, 40% RPMI 1640 medium.

Populations of interest for use in the methods of the invention includesubstantially pure compositions, e.g. at least about 50% HSC, at leastabout 75% HSC, at least about 85% HSC, at least about 95% HSC or more;or may be combinations of one or more stem and progenitor cellspopulations, e.g. white cells obtained from apheresis, etc. Wherepurified cell populations are desired, the target population may bepurified in accordance with known techniques. For example, a populationcontaining white blood cells, particularly including blood or bonemarrow samples, are stained with reagents specific for markers presentof hematopoietic stem and progenitor cells, which markers are sufficientto distinguish the major stem and progenitor groups. The reagents, e.g.antibodies, may be detectably labeled, or may be indirectly labeled inthe staining procedure.

Any combination of markers may be used that are sufficient to select forthe stem/progenitor cells of interest. A marker combination of interestmay include CD34 and CD38, which distinguishes hematopoietic stem cells,(CD34⁺, CD38⁻) from progenitor cells, which are CD34⁺, CD38⁺). HSC arelineage marker negative, and positive for expression of CD90.

In the myeloid lineage are three cell populations, termed CMPs, GMPs,and MEPs. These cells are CD34⁺ CD38⁺, they are negative for multiplemature lineage markers including early lymphoid markers such as CD7,CD10, and IL-7R, and they are further distinguished by the markersCD45RA, an isoform of CD45 that can negatively regulate at least someclasses of cytokine receptor signaling, and IL-3R. These characteristicsare CD45RA⁻ IL-3Rα^(lo) (CMPs), CD45RA⁺IL-3Rα^(lo) (GMPs), and CD45RA⁻IL-3Rα⁻ (MEPs). CD45RA⁻ IL-3Rα^(lo) cells give rise to GMPs and MEPs andat least one third generate both GM and MegE colonies on a single-celllevel. All three of the myeloid lineage progenitors stain negatively forthe markers Thy-1 (CD90), IL-7Rα (CD127); and with a panel of lineagemarkers, which lineage markers may include CD2; CD3; CD4; CD7; CD8;CD10; CD11b; CD14; CD19; CD20; CD56; and glycophorin A (GPA) in humansand CD2; CD3; CD4; CD8; CD19; IgM; Ter110; Gr-1 in mice. With theexception of the mouse MEP subset, all of the progenitor cells are CD34positive. In the mouse all of the progenitor subsets may be furthercharacterized as Sca-1 negative, (Ly-6E and Ly-6A), and c-kit high. Inthe human, all three of the subsets are CD38⁺.

Common lymphoid progenitors, CLP, express low levels of c-kit (CD117) ontheir cell surface. Antibodies that specifically bind c-kit in humans,mice, rats, etc. are known in the art. Alternatively, the c-kit ligand,steel factor (Slf) may be used to identify cells expressing c-kit. TheCLP cells express high levels of the IL-7 receptor alpha chain (CDw127).Antibodies that bind to human or to mouse CDw127 are known in the art.Alternatively, the cells are identified by binding of the ligand to thereceptor, IL-7. Human CLPs express low levels of CD34. Antibodiesspecific for human CD34 are commercially available and well known in theart. See, for example, Chen et al. (1997) Immunol Rev 157:41-51. HumanCLP cells are also characterized as CD38 positive and CD10 positive. TheCLP subset also has the phenotype of lacking expression of lineagespecific markers, exemplified by B220, CD4, CD8, CD3, Gr-1 and Mac-1.The CLP cells are characterized as lacking expression of Thy-1, a markerthat is characteristic of hematopoietic stem cells. The phenotype of theCLP may be further characterized as MeI-14⁻, CD43^(lo), HSA^(lo), CD45⁺and common cytokine receptor y chain positive.

Megakaryocyte progenitor cells (MKP) cells are positive for CD34expression, and tetraspanin CD9 antigen. The CD9 antigen is a 227-aminoacid molecule with 4 hydrophobic domains and 1 N-glycosylation site. Theantigen is widely expressed, but is not present on certain progenitorcells in the hematopoietic lineages. The MKP cells express CD41, alsoreferred to as the glycoprotein IIb/IIIa integrin, which is the plateletreceptor for fibrinogen and several other extracellular matrixmolecules, for which antibodies are commercially available, for examplefrom BD Biosciences, Pharmingen, San Diego, Calif., catalog number340929, 555466. The MKP cells are positive for expression of CD117,which recognizes the receptor tyrosine kinase c-Kit. Antibodies arecommercially available, for example from BD Biosciences, Pharmingen, SanDiego, Calif., Cat. No. 340529. MKP cells are also lineage negative, andnegative for expression of Thy-1 (CD90).

The phrase “solid tumor” as used herein refers to an abnormal mass oftissue that usually does not contain cysts or liquid areas. Solid tumorsmay be benign or malignant. Different types of solid tumors are namedfor the type of cells that form them. Examples of solid tumors aresarcomas, carcinomas, lymphomas etc.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,used interchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

A “host cell”, as used herein, refers to a microorganism or a eukaryoticcell or cell line cultured as a unicellular entity which can be, or hasbeen, used as a recipient for a recombinant vector or other transferpolynucleotides, and include the progeny of the original cell which hasbeen transfected. It is understood that the progeny of a single cell maynot necessarily be completely identical in morphology or in genomic ortotal DNA complement as the original parent, due to natural, accidental,or deliberate mutation.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, “leukemia” areused interchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation. Ingeneral, cells of interest for detection or treatment in the presentapplication include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Detection ofcancerous cells is of particular interest. The term “normal” as used inthe context of “normal cell,” is meant to refer to a cell of anuntransformed phenotype or exhibiting a morphology of a non-transformedcell of the tissue type being examined. “Cancerous phenotype” generallyrefers to any of a variety of biological phenomena that arecharacteristic of a cancerous cell, which phenomena can vary with thetype of cancer. The cancerous phenotype is generally identified byabnormalities in, for example, cell growth or proliferation (e.g.,uncontrolled growth or proliferation), regulation of the cell cycle,cell mobility, cell-cell interaction, or metastasis, etc.

“Therapeutic target” refers to a gene or gene product that, uponmodulation of its activity (e.g., by modulation of expression,biological activity, and the like), can provide for modulation of thecancerous phenotype. As used throughout, “modulation” is meant to referto an increase or a decrease in the indicated phenomenon (e.g.,modulation of a biological activity refers to an increase in abiological activity or a decrease in a biological activity).

Diagnosis and Imaging of Cancer

Detection of calreticulin expression, e.g. cell surface protein, mRNA,etc., particularly cell surface protein, is used alone or in conjunctionwith CD47 expression for clinical diagnostic applications includingprimary diagnosis of cancers, monitoring of interval diseaseprogression, and monitoring of minimal residual disease status,including without limitation hematopoietic malignancies including acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), acutelymphocytic leukemia (ALL); and non-Hodgkin lymphoma (NHL) as well assolid tumors including but not limited to bladder, ovarian, braincancers, and other epithelial cancers. Cancer stem cells of interestalso include carcinomas, e.g. squamous cell carcinoma, ovariancarcinoma, etc.; glioblastomas, and the like. Of interest is includedthe detection and treatment of cells prior to chemotherapeutictreatment.

In a related embodiment, an agent that selectively binds to CRT, e.g.soluble LRP, anti-CTR antibody, etc. is labeled with a detectablemoiety, e.g. a fluorophore, imaging radioisotype, etc. for clinicaldiagnostic imaging applications including primary diagnosis of cancers,monitoring of interval disease progression, and monitoring of minimalresidual disease status. Imaging may be performed in vivo or ex vivo.

Detection of CTR expression is also used in prognosis of cancer, whereincreased levels of CTR are shown to be associated with a worse clinicalprognosis in multiple human malignancies.

Of particular interest is the detection of CRT expression on cancer stemcells, where it has been found that CRT expression segregates withtumorigenicity. Expression of CRT is used alone or in combination withother cancer stem cell markers, e.g. CD47, CD44, etc. to identify,target and/or isolated cancer stem cells.

Binding agents specific for CRT, e.g. antibodies, may be utilized forimmunophenotyping of cells and biological samples. Monoclonal antibodiesdirected against a specific epitope, or combination of epitopes, willallow for the screening of cellular populations expressing the CRT.Various techniques can be utilized using monoclonal antibodies to screenfor cellular populations expressing CRT, and include magnetic separationusing antibody-coated magnetic beads, “panning” with antibody attachedto a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S.Pat. No. 5,985,660; and Morrison et al. Cell, 96:737-49 (1999)). Thesetechniques allow for the screening of particular populations of cells;in immunohistochemistry of biopsy samples; in detecting the presence ofmarkers shed by cancer cells into the blood and other biologic fluids,and the like.

The presence of CRT in a patient sample can be indicative of the stageof the cancer. In addition, detection of CRT can be used to monitorresponse to therapy and to aid in prognosis. The presence of CRT can beutilized for quantitating the cells having the phenotype of the stemcell. In addition to cell surface phenotyping, it may be useful toquantitate the cells in a sample that have a “stem cell” character,which may be determined by functional criteria, such as the ability toself-renew, to give rise to tumors in vivo, e.g. in a xenograft model,and the like.

Clinical samples for use in the methods of the invention may be obtainedfrom a variety of sources, particularly blood and tumor biopsy samples,although in some instances samples such as bone marrow, lymph,cerebrospinal fluid, synovial fluid, and the like may be used. Suchsamples can be separated by centrifugation, elutriation, densitygradient separation, apheresis, affinity selection, panning, FACS,centrifugation with Hypaque, etc. prior to analysis, and usually amononuclear fraction (PBMC) will be used. Once a sample is obtained, itcan be used directly, frozen, or maintained in appropriate culturemedium for short periods of time. Various media can be employed tomaintain cells. The samples may be obtained by any convenient procedure,such as the drawing of blood, venipuncture, biopsy, or the like. Usuallya sample will comprise at least about 10² cells, more usually at leastabout 10³ cells, and preferable 10⁴, 10⁵ or more cells. Typically thesamples will be from human patients, although animal models may finduse, e.g. equine, bovine, porcine, canine, feline, rodent, e.g. mice,rats, hamster, primate, etc.

An appropriate solution may be used for dispersion or suspension of thecell sample. Such solution will generally be a balanced salt solution,e.g. normal saline, PBS, Hank's balanced salt solution, etc.,conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc.

Analysis of the cell staining will use conventional methods. Techniquesproviding accurate enumeration include fluorescence activated cellsorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. The cells may be selected againstdead cells by employing dyes associated with dead cells (e.g. propidiumiodide).

The CRT affinity reagents may be antibodies, specific receptors orligands for the cell surface molecules indicated above. In addition toantibody reagents, peptide-MHC antigen and T cell receptor pairs may beused; peptide ligands and receptors; effector and receptor molecules,and the like. Antibodies and T cell receptors may be monoclonal orpolyclonal, and may be produced by transgenic animals, immunizedanimals, immortalized human or animal B-cells, cells transfected withDNA vectors encoding the antibody or T cell receptor, etc. The detailsof the preparation of antibodies and their suitability for use asspecific binding members are well-known to those skilled in the art.

The antibodies are added to a suspension of cells, and incubated for aperiod of time sufficient to bind the available cell surface antigens.The incubation will usually be at least about 5 minutes and usually lessthan about 30 minutes. It is desirable to have a sufficientconcentration of antibodies in the reaction mixture, such that theefficiency of the separation is not limited by lack of antibody. Theappropriate concentration is determined by titration. The medium inwhich the cells are separated will be any medium that maintains theviability of the cells. A preferred medium is phosphate buffered salinecontaining from 0.1 to 0.5% BSA. Various media are commerciallyavailable and may be used according to the nature of the cells,including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic SaltSolution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI,Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplemented withfetal calf serum, BSA, HSA, etc.

The labeled cells are then quantitated as to the expression of cellsurface markers. The comparison of a differential analysis obtained froma patient sample, and a reference differential progenitor analysis isaccomplished by the use of suitable deduction protocols, Al systems,statistical comparisons, etc. A comparison with a reference differentialprogenitor analysis from normal cells, cells from similarly diseasedtissue, and the like, can provide an indication of the disease staging.A database of reference differential progenitor analyses can becompiled. An analysis of particular interest tracks a patient, e.g. atinitial diagnosis of cancer, following therapy, etc. The methods of theinvention allow early therapeutic intervention, e.g. initiation ofchemotherapy or antibody mediated therapy, increase of dose, changingselection of drugs, and the like.

In another embodiment, methods are provided for targeting or depletingcancer stem cells, the method comprising contacting a population ofcells, e.g. blood from a cancer patient, with a reagent thatspecifically binds CTR in order to target or deplete the cancer stemcells. In certain aspects, the reagent is an antibody conjugated to acytotoxic agent, e.g. radioactive isotope, chemotherapeutic agent,toxin, etc. In some embodiments, the depletion is performed on an exvivo population of cells, e.g. the purging of autologous stem cellproducts (mobilized peripheral blood or bone marrow) for use inautologous transplantation for cancer patients.

In some embodiments of the invention, the number of cancer stem cells(CSC), which express CRT, in a patient sample is determined relative tothe total number of cancer cells, where a greater percentage of CSC isindicative of the potential for continued self-renewal of cells with thecancer phenotype. The quantitation involves detection of CRT expression,and may further utilize expression of other markers known to be found onCSC, including upregulated CD47, CD44, markers of AML and CML LSC, andthe like. The quantitation of CSC in a patient sample may be compared toa reference population, e.g. a patient sample such as a blood sample, aremission patient sample, etc. In some embodiments, the quantitation ofCSC is performed during the course of treatment, where the number ofcancer cells and the percentage of such cells that are CSC arequantitated before, during and as follow-up to a course of therapy.Desirably, therapy targeted to cancer stem cells results in a decreasein the total number, and/or percentage of CSC in a patient sample.

In other embodiments of the invention, anti-cancer agents are targetedto CSC by specific binding to CRT, or a combination of CRT and a secondmarker, including CD47. In such embodiments, the anti-cancer agentsinclude antibodies and antigen-binding derivatives thereof specific fora marker or combination of markers of the present invention, which areoptionally conjugated to a cytotoxic moiety. Depletion of CSC istherapeutically useful. Depletion achieves a reduction in circulatingCSC by up to about 30%, or up to about 40%, or up to about 50%, or up toabout 75% or more. Depletion can be achieved by using an agent todeplete CSC either in vivo or ex vivo.

The CSC are identified by their phenotype with respect to particularmarkers, and/or by their functional phenotype. In some embodiments, theCSC are identified and/or isolated by binding to the cell with reagentsspecific for the markers of interest. The cells to be analyzed may beviable cells, or may be fixed or embedded cells. In some embodiments,the reagents specific for the markers of interest are antibodies, whichmay be directly or indirectly labeled. Such antibodies will usuallyinclude antibodies specific for a marker or combination of markers ofthe present invention.

A cancer, including without limitation AML, ALL, CML, etc. can be stagedby analysis of the presence of cancer stem cells. Staging is useful forprognosis and treatment. In one embodiment of the invention, a samplefrom a leukemia patient is stained with reagents specific for a markeror combination of markers of the present invention. The analysis ofstaining patterns provides the relative distribution of CSC, whichdistribution predicts the stage of leukemia. In some embodiments, thesample is analyzed by histochemistry, including immunohistochemistry, insitu hybridization, immunofluorescence and the like, for the presence ofCD34⁺CD38⁻ cells that express a marker or combination of markers of thepresent invention. The presence of such cells indicates the presence ofCSC. In one embodiment, the patient sample is compared to a control, ora standard test value. In another embodiment, the patient sample iscompared to a pre-leukemia sample, or to one or more time points throughthe course of the disease. Samples, including tissue sections, slides,etc. are stained with reagents specific for markers that indicate thepresence of cancer stem cells. Samples may be frozen, embedded, presentin a tissue microarray, and the like. The reagents, e.g. antibodies,polynucleotide probes, etc. may be detectably labeled, or may beindirectly labeled in the staining procedure.

The information thus derived is useful in prognosis and diagnosis,including susceptibility to acceleration of disease, status of adiseased state and response to changes in the environment, such as thepassage of time, treatment with drugs or other modalities. The cells canalso be classified as to their ability to respond to therapeutic agentsand treatments, isolated for research purposes, screened for geneexpression, and the like. The clinical samples can be furthercharacterized by genetic analysis, proteomics, cell surface staining, orother means, in order to determine the presence of markers that areuseful in classification. For example, genetic abnormalities can becausative of disease susceptibility or drug responsiveness, or can belinked to such phenotypes.

Depletion of CSC is useful in the treatment of cancer. Depletion can beachieved by several methods. Depletion is defined as a reduction in thetarget population by up to about 30%, or up to about 40%, or up to about50%, or up to about 75% or more. An effective depletion is usuallydetermined by the sensitivity of the particular disease condition to thelevels of the target population. Thus in the treatment of certainconditions a depletion of even about 20% could be beneficial.

A CRT specific agent that specifically depletes the targeted CSC is usedto contact the patient in vitro or in vivo, wherein after the contactingstep, there is a reduction in the number of viable CSC in the targetedpopulation. An effective dose of antibodies for such a purpose issufficient to decrease the targeted population to the desired level, forexample as described above. Antibodies for such purposes may have lowantigenicity in humans or may be humanized antibodies.

In one embodiment of the invention, antibodies for depleting targetpopulation are added to patient blood in vivo. In another embodiment,the antibodies are added to the patient blood ex vivo. Beads coated withthe antibody of interest can be added to the blood, target cells boundto these beads can then be removed from the blood using procedurescommon in the art. In one embodiment the beads are magnetic and areremoved using a magnet. Alternatively, when the antibody isbiotinylated, it is also possible to indirectly immobilize the antibodyonto a solid phase which has adsorbed avidin, streptavidin, or the like.The solid phase, usually agarose or sepharose beads are separated fromthe blood by brief centrifugation. Multiple methods for taggingantibodies and removing such antibodies and any cells bound to theantibodies are routine in the art. Once the desired degree of depletionhas been achieved, the blood is returned to the patient. Depletion oftarget cells ex vivo decreases the side effects such as infusionreactions associated with the intravenous administration. An additionaladvantage is that the repertoire of available antibodies is expandedsignificantly as this procedure does not have to be limited toantibodies with low antigenicity in humans or humanized antibodies.

In vitro, CSC may be separated from a complex mixture of cells bytechniques that enrich for cells that differentially express CRT orcombination of markers of the present invention. For isolation of cellsfrom tissue, an appropriate solution may be used for dispersion orsuspension. Such solution will generally be a balanced salt solution,e.g. normal saline, PBS, Hank's balanced salt solution, etc.,conveniently supplemented with fetal calf serum or other naturallyoccurring factors, in conjunction with an acceptable buffer at lowconcentration, generally from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc.

The separated cells may be collected in any appropriate medium thatmaintains the viability of the cells, usually having a cushion of serumat the bottom of the collection tube. Various media are commerciallyavailable and may be used according to the nature of the cells,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequentlysupplemented with fetal calf serum.

Compositions highly enriched for CSC are achieved in this manner. Thesubject population may be at or about 50% or more of the cellcomposition, and preferably be at or about 75% or more of the cellcomposition, and may be 90% or more. The desired cells are identified bytheir surface phenotype, by the ability to self-renew, ability to formtumors, etc. The enriched cell population may be used immediately, ormay be frozen at liquid nitrogen temperatures and stored for longperiods of time, being thawed and capable of being reused. The cells maybe stored in 10% DMSO, 90% FCS medium. The population of cells enrichedfor CSC may be used in a variety of screening assays and cultures, asdescribed below.

The enriched CSC population may be grown in vitro under various cultureconditions. Culture medium may be liquid or semi-solid, e.g. containingagar, methylcellulose, etc. The cell population may be convenientlysuspended in an appropriate nutrient medium, such as Iscove's modifiedDMEM or RPMI-1640, normally supplemented with fetal calf serum (about5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, andantibiotics, e.g. penicillin and streptomycin.

In some embodiments of the invention, the expression of CRT on cancercells, including without limitation cancer cells prior to treatment witha chemotherapeutic drug, is utilized to enhance killing of the cancercells. Cancer cells can be contacted with an agonist of CRT, e.g. anagonistic antibody, particularly one that activates LRP, in the presenceof phagocytic cells in order to enhance phagocytosis of the cancercells. Cancer cells can also be contacted with an antibody to CRT thateliminates cancer cells by ADCC, complement, and other Fc-receptormediated effects. In some such embodiments, the CRT agonist isadministered in combination with an agent that blocks CD47 signaling,e.g. soluble SIRPa, anti-CD47, and the like. Included in such agents arebi-specific antibodies targeted to both CD47 and CRT or CD47 and LRP.Also included are agents comprising a CD47 blocking moiety and an activeportion of CTR protein.

In related embodiments, cancer cells, including without limitationcancer cells prior to treatment with a chemotherapeutic drug, arecontacted with an agent that selectively binds to CRT, includingantibodies, soluble LRP, etc., which agent is optionally conjugated to atoxic moiety, e.g. a radionuclide, toxin, etc. to induce killing of thecell to which the agent has bound.

“Reducing growth of cancer cells” includes, but is not limited to,reducing proliferation of cancer cells, and reducing the incidence of anon-cancerous cell becoming a cancerous cell. Whether a reduction incancer cell growth has been achieved can be readily determined using anyknown assay, including, but not limited to, [³H]-thymidineincorporation; counting cell number over a period of time; detectingand/or measuring a marker associated with the cancer, etc.

Whether a substance, or a specific amount of the substance, is effectivein treating cancer can be assessed using any of a variety of knowndiagnostic assays for cancer, including, but not limited to biopsy,contrast radiographic studies, CAT scan, and detection of a tumor markerassociated with cancer in the blood of the individual. The substance canbe administered systemically or locally, usually systemically.

As an alternative embodiment, an agent, e.g. a chemotherapeutic drugthat reduces cancer cell growth, can be targeted to a cancer cell byconjugation to a CTR specific antibody. Thus, in some embodiments, theinvention provides a method of delivering a drug to a cancer cell,comprising administering a drug-antibody complex to a subject, whereinthe antibody is specific for CTR, and the drug is one that reducescancer cell growth, a variety of which are known in the art. Targetingcan be accomplished by coupling (e.g., linking, directly or via a linkermolecule, either covalently or non-covalently, so as to form adrug-antibody complex) a drug to an antibody specific for acancer-associated polypeptide.

In certain embodiments, a bi-specific antibody may be used. For examplea bi-specific antibody in which one antigen binding domain is directedagainst CTR and the other antigen binding domain is directed against acancer cell marker, such as CD47, CD96, CD97, CD99, PTHR2, HAVCR2 etc.

For administration, the antibody-therapeutic or antibody-imaging agentwill generally be mixed, prior to administration, with a non-toxic,pharmaceutically acceptable carrier substance. Usually, this will be anaqueous solution, such as normal saline or phosphate-buffered saline(PBS), Ringer's solution, lactate-Ringer's solution, or any isotonicphysiologically acceptable solution for administration by the chosenmeans. Preferably, the solution is sterile and pyrogen-free, and ismanufactured and packaged under current Good Manufacturing Processes(GMPs), as approved by the FDA. The clinician of ordinary skill isfamiliar with appropriate ranges for pH, tonicity, and additives orpreservatives when formulating pharmaceutical compositions foradministration by intravascular injection, intrathecal injection,injection into the cerebro-spinal fluid, direct injection into thetumor, or by other routes. In addition to additives for adjusting pH ortonicity, the antibody-therapeutics and antibody-imaging agents may bestabilized against aggregation and polymerization with amino acids andnon-ionic detergents, polysorbate, and polyethylene glycol. Optionally,additional stabilizers may include various physiologically-acceptablecarbohydrates and salts. Also, polyvinylpyrrolidone may be added inaddition to the amino acid. Suitable therapeutic immunoglobulinsolutions which are stabilized for storage and administration to humansare described in U.S. Pat. No. 5,945,098, incorporated fully herein byreference. Other agents, such as human serum albumin (HSA), may be addedto the therapeutic or imaging composition to stabilize the antibodyconjugates.

The compositions of the invention may be administered using anymedically appropriate procedure, e.g., intravascular (intravenous,intraarterial, intracapillary) administration, injection into thecerebrospinal fluid, intracavity, subcutaneously, or direct injection inthe tumor. For the imaging compositions of the invention, administrationvia intravascular injection is preferred for pre-operative visualizationof the tumor.

The effective amount of the therapeutic antibody-conjugate compositionor of the imaging antibody-conjugate compositions to be given to aparticular patient will depend on a variety of factors, several of whichwill be different from patient to patient. A competent clinician will beable to determine an effective amount of a therapeuticantibody-conjugate composition to administer to a patient to retard thegrowth and promote the death of tumor cells, or an effective amount ofan imaging composition to administer to a patient to facilitate thevisualization of a tumor. Dosage of the antibody-conjugate will dependon the treatment of the tumor, route of administration, the nature ofthe therapeutics, sensitivity of the tumor to the therapeutics, etc.Utilizing LD₅₀ animal data, and other information available for theconjugated cytotoxic or imaging moiety, a clinician can determine themaximum safe dose for an individual, depending on the route ofadministration. For instance, an intravenously administered dose may bemore than an intrathecally administered dose, given the greater body offluid into which the therapeutic composition is being administered.Similarly, compositions which are rapidly cleared from the body may beadministered at higher doses, or in repeated doses, in order to maintaina therapeutic concentration. Imaging moieties are typically less toxicthan cytotoxic moieties and may be administered in higher doses in someembodiments. Utilizing ordinary skill, the competent clinician will beable to optimize the dosage of a particular therapeutic or imagingcomposition in the course of routine clinical trials.

Typically the dosage will be 0.001 to 100 milligrams of conjugate perkilogram subject body weight. Doses in the range of 0.01 to 1 mg perkilogram of patient body weight may be utilized for a radionuclidetherapeutic composition which is administered intrathecally. Relativelylarge doses, in the range of 0.1 to 10 mg per kilogram of patient bodyweight, may used for imaging conjugates with a relatively non-toxicimaging moiety. The amount utilized will depend on the sensitivity ofthe imaging method, and the relative toxicity of the imaging moiety. Ina therapeutic example, where the therapeutic composition comprises a¹³¹I cytotoxic moiety, the dosage to the patient will typically start ata lower range of 10 mCi, and go up to 100, 300 or even 500 mCi. Statedotherwise, where the therapeutic agent is ¹³¹I, the dosage to thepatient will typically be from 5,000 Rads to 100,000 Rads (preferably atleast 13,000 Rads, or even at least 50,000 Rads). Doses for otherradionuclides are typically selected so that the tumoricidal dose willbe equivalent to the foregoing range for ¹³¹I. Similarly, chemotoxic ortoxin protein doses may be scaled accordingly.

The antibody conjugate can be administered to the subject in a series ofmore than one administration. For therapeutic compositions, regularperiodic administration (e.g., every 2-3 days) will sometimes berequired, or may be desirable to reduce toxicity. For therapeuticcompositions which will be utilized in repeated-dose regimens, antibodymoieties which do not provoke HAMA or other immune responses arepreferred. The imaging antibody conjugate compositions may beadministered at an appropriate time before the visualization technique.For example, administration within an hour before direct visualinspection may be appropriate, or administration within twelve hoursbefore an MRI scan may be appropriate. Care should be taken, however, tonot allow too much time to pass between administration andvisualization, as the imaging compound may eventually be cleared fromthe patient's system.

Methods for Transplantation

In other therapeutic methods, hematopoietic cells, including withoutlimitation HSC, hematopoietic progenitors, normal bone marrow, ormobilized peripheral blood for patients with a clinical indication forhematopoietic transplantation, are protected from phagocytosis incirculation by providing a host animal with an agent that blocks theinteraction between CRT and LRP, e.g. an antibody selective for CRT, anantibody selective for LRP, soluble CRT or LRP, a CRT blocking peptide,and the like, is administered, which blocks the pro-phagocytic signaland decreases the clearance of the hematopoietic cells from circulation.In some embodiments of the invention, the agent, e.g. peptide, solubleCRT, etc. is provided as a fusion protein, for example fused to an Fcfragment, e.g., IgG1 Fc, IgG2 Fc, Ig A Fc etc.

The subject invention provide for methods for transplantinghematopoietic cells into a mammalian recipient. A need fortransplantation may be caused by genetic or environmental conditions,e.g. chemotherapy, exposure to radiation, etc. The cells fortransplantation may be mixtures of cells, e.g. buffy coat lymphocytesfrom a donor, or may be partially or substantially pure. The cells maybe autologous cells, particularly if removed prior to cytoreductive orother therapy, or allogeneic cells, and may be used for hematopoieticstem or progenitor cell isolation and subsequent transplantation.

The cells may be combined with calreticulin blocking agent, including ablocking peptide, blocking oligosaccharide, blocking antibody, etc.prior to transplantation or transfusion. For example, the cells may becombined with the blocking agent at a concentration of from about 10μg/ml, about 100 μg/ml, about 1 mg/ml, about 10 mg/ml, etc., at atemperature of from about 4°, about 10°, about 25° about 37°, for aperiod of time sufficient to coat the cells, where in some embodimentsthe cells are maintained on ice. In other embodiments the cells arecontacted with the CTR blocking agent immediately prior to introductioninto the recipient, where the concentrations are as described above.

The composition comprising hematopoietic cells and a CTR blocking agentis administered in any physiologically acceptable medium, normallyintravascularly, although they may also be introduced into bone or otherconvenient site, where the cells may find an appropriate site forregeneration and differentiation. Usually, at least 1×10⁵ cells will beadministered, preferably 1×10⁶ or more. The composition may beintroduced by injection, catheter, or the like.

Example 1 Therapeutic and Diagnostic Methods for ManipulatingPhagocytosis Through Calreticulin and Low Density Lipoprotein-RelatedReceptor

Cell Surface Calreticulin is Expressed on Cancer, but not Most Normal,Stem and Progenitor Cells.

Cell surface calreticulin expression was determined on a variety ofprimary human cancer cells and their normal cell counterparts by flowcytometry. In hematologic malignancies, cell surface calreticulin wasexpressed on a greater percentage of bulk cells in AML (average=23.9%),acute lymphocytic leukemia (ALL, 17.6%), chronic phase chronic myeloidleukemia (CML, 47.6%), and NHL (18.3%) when compared to normal bonemarrow (2.6%) and normal peripheral blood cells (2.6%) (FIG. 1A). Insolid tumors, cell surface calreticulin was also expressed on a greaterpercentage of bulk cells in ovarian cancer (average=20.5%), glioblastoma(31.7%), and bladder cancer (23.7%) when compared to normal fetalneurons (0.3%), astrocytes, (2.5%) and normal fetal bladder cells(1.41%) (FIG. 1B). In this analysis, annexin V-positive cells wereexcluded, indicating that calreticulin-positive cancer cells were not apart of the apoptotic subset. In addition, calreticulin positive-cancercells (from AML and bladder cancer patients) formed tumors whenengrafted into immunodeficient mice similarly to CRT-negative cancercells, indicating that CRT-positive cancer cells were functionallyviable and possess tumorigenic potential in vivo (FIG. 5).

Previous studies have identified that the endoplasmic reticulum (ER)protein ERp57 co-translocates with CRT to the cell surface and isrequired for CRT cell surface exposure under conditions of apoptosis.Accordingly, we assessed the relationship between cell surface CRT andERp57 expression on tumor cells. On non-apoptotic (annexin V negative)tumor cells, cell surface ERp57 expression was associated with cellsurface CRT expression (FIG. 6A). Furthermore, across several differenttumor types (including primary human tumor samples and cancer celllines), ERp57 was expressed on a higher percentage of CRT+ cellscompared to CRT-counterparts (FIG. 6B,C).

Given that primary human tumors are heterogeneous and contain asubpopulation of tumor-initiating cells, we next investigated whethercell surface calreticulin was present on the cancer stem cell (CSC)population of each tumor type in which the immunophenotype of functionalCSC is known. In AML and chronic phase CML, cell surface calreticulinwas expressed on CD34+CD38-CD90-Lin− AML (19, 20) andCD34+CD38-CD90+chronic phase CML leukemia stem cells (LSC), as well asdownstream progenitor populations, while normal bone marrowhematopoietic stem and progenitor populations expressed minimal cellsurface calreticulin (FIG. 1C,D). For AML, similar levels of cellsurface calreticulin expression were observed for LSC compared to othercellular subsets (FIG. 10). In contrast, CML LSC expressed higher levelsof cell surface calreticulin compared to downstream CMP and GMPpopulations (FIG. 1D). Cell surface calreticulin was also expressed onCSC of solid tumors including CD44+Lin− bladder CSC and CD133+Lin−glioblastoma CSC (22, 23) (FIG. 1E).

We next determined whether there was a correlation between calreticulin(CRT) and CD47 expression in human tissues, postulating that a balancebetween pro-(CRT) and anti-(CD47) phagocytic signals may be maintainedas a homeostatic mechanism. CRT and CD47 cell surface expression wereprofiled in a variety of human cancer cell lines, primary cancers, andnormal cells. CD47 expression correlated with CRT expression in avariety of hematologic and solid tumor cell lines as well as in primaryhuman AML, CML, and ALL patient samples (FIG. 2A). Notably, normal cellsexpressed minimal levels of both CRT and CD47 (FIG. 2A, top panels). Innormal human bone marrow and fetal bladder, those cells that were CRTpositive expressed higher levels of CD47 compared to CRT negativecellular counterparts (FIG. 7). Thus, in both normal and cancer cells,there is a strong positive correlation between CRT and CD47 expression.

Increased CD47 on Cancer Cells Protects them from Calreticulin-MediatedPhagocytosis.

We observed increased cell surface calreticulin and CD47 on human cancercells leading us to hypothesize that increased CD47 protects these cellsfrom calreticulin-mediated phagocytosis. To investigate this hypothesis,we performed in vitro phagocytosis assays on two differentCRT-expressing cancer cell lines: one expressing high CD47 levels (Raji)and one deficient in CD47 expression (MOLM13). First, Raji cells, aBurkitt's NHL cell line that expresses high levels of CD47 andcalreticulin (FIG. 2B and FIG. 8), were incubated with human macrophagesunder conditions where CD47 expression was knocked down to variouslevels by lentiviral transduction of shRNAs (FIG. 2B,C). Cell surfacecalreticulin expression was unaffected by shRNA-mediated CD47 knockdown(FIG. 8). Upon incubation with human macrophages, Raji cells withapproximately 2 fold knockdown of CD47 expression (shCD47-1 andshCD47-2) were more robustly phagocytosed by human macrophages than werewild type and GAPD control transduced Raji cells which were theminimally phagocytosed (FIG. 2D). Phagocytosis of shCD47-1 and shCD47-2Raji cells was dependent on the calreticulin-LRP interaction as theobserved phagocytosis was completely abrogated in the presence of a CRTblocking peptide (FIG. 2D). In the second experiment, MOLM13 cells, ahuman AML cell line that is deficient in CD47 expression but expressescalreticulin (FIG. 8), were incubated with human macrophages. Asexpected, MOLM13 cells were robustly phagocytosed at baseline, whilephagocytosis was significantly reduced when the CRT-LRP interaction wasblocked (FIG. 2E). These findings demonstrate that overexpression ofCD47 in cancers counterbalances calreticulin-mediated phagocytosis.

Calreticulin is the dominant pro-phagocytic signal on several humancancers and is required for anti-CD47 antibody-mediated phagocytosis. Inprior studies, we demonstrated that in several human cancersoverexpression of CD47 contributes to evasion of macrophagephagocytosis, and furthermore that monoclonal antibody-mediated blockadeof CD47 can enable phagocytosis and elimination of tumors in vitro andin mouse xenografts. However, we also showed that normal hematopoieticprogenitor cells, which express CD47, were not phagocytosed when coatedwith anti-CD47 antibody. Additionally, administration of a blockinganti-mouse CD47 antibody to wild type mice caused minimal tissuetoxicity. The lack of antibody toxicity is not likely exclusively due tooverexpression of CD47 on cancer cells compared to normal counterpartsgiven that both normal and cancer cells are coated with anti-CD47antibody at therapeutic doses. Instead, it is likely a result of thefact that, in order for target cells to be phagocytosed upon blockade ofan anti-phagocytic signal (CD47), the cells must also display a potentpro-phagocytic signal, which is absent on normal cells.

Given the known role of CRT as a pro-phagocytic signal, its correlationwith CD47 expression (FIG. 2A), and its ability to be counteracted byCD47 (FIG. 2), we investigated whether the expression of cell surfaceCRT on cancer but not normal cells could explain the selective targetingof tumor cells by a blocking anti-CD47 antibody. In vitro phagocytosisassays were performed by incubating primary human normal cells or cancercells with human macrophages in the presence of anti-CD47 antibody. CD47was expressed on all normal and cancer cells profiled (FIG. 2A and FIG.7, 9), but expression of calreticulin was primarily restricted to tumorcells (FIG. 1A,B). No phagocytosis of cells from a variety of normalhuman tissue types was observed with anti-CD47 antibody (FIG. 3B), whileprimary cancer cells from a variety of tumor types were robustlyphagocytosed (FIG. 3A,C). Significantly, anti-CD47 antibody-mediatedphagocytosis of cancer cells was completely abrogated in most cases whencells were simultaneously incubated with peptides that inhibit theCRT-LRP interaction, including a calreticulin blocking peptide andreceptor-associated protein (RAP), an inhibitor of LRP (see Gardai etal. Cell. 123, 321-334 (2005), herein specifically incorporated byreference). (FIG. 3C).

Increasing concentrations of a calreticulin blocking peptide lead to adose-dependent reduction in anti-CD47 antibody mediated phagocytosis(FIG. 10). Notably, additional blockade of other pro-phagocytic signalswas not required to abolish anti-CD47 antibody-mediated phagocytosis, ascells incubated with anti-CD47 antibody under CRT-LRP blockade werephagocytosed at levels similar to baseline controls (FIG. 3C). However,two bladder cancer samples exhibited higher baseline levels ofphagocytosis with IgG1 isotype control compared to other cancer celltypes which may be due to expression of other pro-phagocytic signals onthese specific cells. Nevertheless, blockade of CRT or LRP in thepresence of anti-CD47 antibody abrogated phagocytosis of these bladdercancer cells to levels similar to IgG1 isotype controls. Blockade of thecalreticulin-LRP interaction alone by CRT blocking peptide or LRP had noeffect on phagocytosis when compared to IgG control (FIG. 3C).

Next, the relationship between the level of tumor cell surface CRTexpression and level of phagocytosis by anti-CD47 antibody wasinvestigated. Cell surface CRT expression on tumor cells positivelycorrelated with the degree of anti-CD47 antibody mediated phagocytosis,regardless of tumor cell type (FIG. 3D). Finally, given that normalcells express minimal levels of cell surface CRT, we investigatedwhether the addition of CRT to the surface of these cells could enablephagocytosis. An in vitro phagocytosis assay was performed on NBM cellsincubated with exogenous recombinant calreticulin protein, previouslydemonstrated to adsorb onto the cellular surface and directly bind LRP.In contrast to vehicle control, incubation with exogenous CRT enabledphagocytosis of NBM cells while anti-CD47 antibody did not (FIG. 3E).Collectively, these results demonstrate that anti-CD47 antibody-mediatedphagocytosis requires the presence of cell surface calreticulin.

Increased Calreticulin Expression Confers a Worse Clinical Prognosis inMultiple Human Malignancies.

Lastly, we sought to investigate the clinical relevance of thesefindings by investigating the association between CRT expression andclinical outcomes. We analyzed calreticulin mRNA levels in patients withhuman malignancies of distinct tumor types and investigated theircorrelation with tumor progression and clinical outcome. Utilizingpreviously published gene profiling datasets with associated clinicaloutcome data, we determined calreticulin expression in both hematologicand solid tumor malignancies, including non-Hodgkin lymphoma (mantlecell lymphoma (MCL)), superficial and invasive bladder cancer, andneuroblastoma. Patients were stratified into calreticulin high andlow-expressing cohorts relative to the median value and analyzed forclinical outcomes. For each tumor type, correlations betweencalreticulin expression and event-free, disease-specific, or overallsurvival were measured in two independent datasets to test and validatesignificant associations. Regardless of tumor type, higher calreticulinexpression predicted a worse clinical outcome in all malignanciesanalyzed: neuroblastoma (FIG. 4A,B), bladder cancer (FIG. 4C,D), and NHL(MCL, FIG. 4E,F). These associations were significant when calreticulinexpression was considered either as a dichotomous variable (relative tothe median) or as a continuous variable (table 1). The prognostic powerof CRT was independent from type of therapy as patients with the varioustumors received disparate treatments including observation, surgery, orchemotherapy (table 1). Additionally, the prognostic power of CRT waspreserved in both early and late stage tumors as increased calreticulinlevels correlated with worse survival in both superficial and invasivebladder cancer (FIG. 4C.D, table 1). Thus, calreticulin expression isassociated with tumor progression and worse clinical outcome acrossseveral tumor types.

TABLE 1 Table 1: Analysis of the prognostic value of calreticulin inhuman malignancies Summary of statistical analyses is presented fromclinical data in FIG. 4. Dichotomous HR and associated statisticsreflect calreticulin expression cut-off around the median. Statisticsare also presented for calreticulin expression when considered as acontinuous variable with log-likelihood p values within a univariate Coxregression model. Therapy represents all possible therapies administeredwithin each cohort. Disease Data Patients Dichotomous (median)Continuous (dataset) Fig. source (n) Therapy HR 95% CI P value HRZ-score P value Ref Neuroblastoma 4A EBI Array 478  obs, 1.70 1.23-2.35<0.005 2.25 4.07 <0.0001 (1) Express: surgery, L 50 E-MTAB-179 to HR-CX⁺Neuroblastoma 4B EBI Array 251  Obs, surgery, 1.78 1.10-2.87 <0.05 4.783.11 <0.005 51 (2) Express: CTX, VCR, E-TABM-38 cisplatin, DOXSuperficial and 4C NCBI GEO: 165* BCG, radical 2.52 1.17-5.45 <0.05 3.363.48 <0.001 52 invasive bladder GSE135 07 cystectomy/LN cancer (1)dissection, cisplatin- based chemo Invasive 4D NCBI GEO: 30 cisplatin-2.18 0.95-4.98 0.059 2.53 1.99 <0.05 53 bladder cancer (2) GSE5287 basedchemo Mantle cell 4E NCBI GEO: 71 untreated 2.57 1.35-4.89 <0.005 3.023.19 <0.005 54 Lymphoma (1) GSE10793 Mantle cell 4F LLMPP: 92multi-agent 3.06 1.72-5.43 <0.0001 1.72 4.12 <0.0001 55 lymphoma (2)Rosenwald_MCL chemo ⁺first line therapy. *91 patients in this datasethad missing clinical data. Ref = reference, HR = hazard ratio, CI =confidence interval, obs = observation, L to HR-CX = low (<2 cycles) tohigh dose (≧6 cycles) cytotoxic therapy, BCG = bacillus calmette-guerinimmunotherapy, CTX = cyclophosphamide, VCR = vincristine, DOX =doxorubicin, LN = lymph node. In this report, we identify calreticulinas a pro-phagocytic signal highly expressed on the surface of severalhuman cancers, but minimally expressed on normal cell counterparts, anddemonstrate that CRT expression is required for anti-CD47antibody-mediated phagocytosis.

Anti-CD47 Antibody Preferentially Eliminates Tumor Cells Because ofDifferential Expression of Cell Surface Calreticulin.

We recently demonstrated that several cancers overexpress CD47 and thata blocking anti-CD47 monoclonal antibody can eliminate tumor cells invitro and in vivo. These pre-clinical findings provide a strongrationale for the use of an anti-CD47 antibody in the treatment of humancancers. However, given the broad low level expression of CD47 on bothhematopoietic and most other normal tissues, antibody toxicity could bea significant barrier to clinical translation.

To investigate this issue, we previously injected a blocking anti-mouseCD47 antibody into wild type mice at a dose that coated >98% of bonemarrow cells but observed no overt toxicity, with the exception ofisolated neutropenia. Moreover, a recent report demonstrated thatinhibition of CD47 with either an antibody or morpholino could conferradioprotective effects to normal tissues. Here, we demonstrate that,despite low level CD47 expression, normal human cells from severaltissues are not phagocytosed by human macrophages when coated withanti-CD47 antibody (FIG. 3B). We speculate that the selectivephagocytosis of tumor cells is not simply dictated by CD47 expressionlevel, but is also governed by the presence of the pro-phagocytic signalcalreticulin, which is present on tumor cells but not on normal cells.

Several lines of evidence support this hypothesis. First, normal cellsthat express CD47 but not calreticulin are not phagocytosed with ananti-CD47 antibody despite being coated with the antibody (FIG. 3B).Second, tumor cells that express CD47 and calreticulin are phagocytosedwhen coated with anti-CD47 antibody (FIG. 3A,C). Third, phagocytosis oftumor cells with anti-CD47 antibody is completely abrogated when thecalreticulin-LRP interaction is blocked (FIG. 3A,C). Fourth, adsorptionof exogenous CRT onto the surface of NBM cells, which express minimalCRT (FIG. 1A), enabled increased phagocytosis compared to vehiclecontrol or anti-CD47 antibody administration (FIG. 3E). Collectively,these findings demonstrate that calreticulin is necessary for anti-CD47antibody-mediated phagocytosis, and that surface expression of thisprotein is primarily restricted to tumor cells.

This study indicates that the therapeutic window for anti-CD47 antibodytherapy is not just a consequence of CD47 level on target cells, butthat it also depends on the surface expression of pro-phagocyticcalreticulin. On the basis of our findings, the overall contribution ofpro (CRT)- and anti (CD47)-phagocytic signals determines whether normalor tumor cells are phagocytosed at steady state, or by anti-CD47antibody therapy (FIG. 11). At steady state, tumor cells expresscalreticulin, but evade phagocytosis through overexpression of CD47,indicating the dominance of the “don't eat me” anti-phagocytic signal(FIG. 11A,B). Normal cells express low levels of CD47, and avoidphagocytosis because of a lack of CRT expression. In contrast, cellsundergoing DNA damage or apoptosis express calreticulin on their cellsurface, which is dominant over low CD47 expression and leads tophagocytosis. In the context of anti-CD47 antibody therapy, theanti-phagocytic signal (CD47) is blocked, unmasking the pro-phagocyticsignal (CRT) on tumor cells, leading to phagocytosis (FIG. 11C,D). Incontrast, blockade of CD47 on normal cells does not lead to phagocytosissince the pro-phagocytic “eat me” signal (CRT) is absent.

Although calreticulin appears to be primarily expressed on the surfaceof apoptotic or malignant cells, prior reports detected surfacecalreticulin on some human normal cells including activated peripheralblood T cells and circulating neutrophils. In addition, a blockingmonoclonal anti-CD47 antibody enhances phagocytosis of apoptoticneutrophils. Interestingly, in our mouse toxicity studies,administration of a blocking anti-mouse CD47 antibody led to selectivedepletion of neutrophils, while other hematopoietic cells wereunaffected. Similar to tumor cells, this selective neutropenic toxicitymay be due to unmasking of calreticulin on neutrophils when the “don'teat me” signal (CD47) is blocked by anti-CD47 antibody. Although mostnormal cells do not express cell surface calreticulin, normal cells mayupregulate calreticulin under certain conditions, including radiationand anthracycline-based chemotherapy as has been shown in some tumortypes. Our findings provide a cautionary note that normal cells mightupregulate calreticulin as a consequence of radiation andchemotherapy-based cancer therapy, and thus combination chemoradiationand anti-CD47 antibody therapy must be tested for potential increasedtoxicity to normal cells.

Calreticulin is the Dominant Pro-Phagocytic Signal on Several HumanCancers.

We demonstrate that several human cancers, including both hematopoieticand solid tumor malignancies, express the pro-phagocytic signalcalreticulin. Known physiologic pro-phagocytic signals have previouslybeen identified in several cancers including phosphatidylserine andannexin-1. However, most of these studies were not performed on primaryhuman patient samples as in this study. Additionally, ligand expressionappears to be mixed across tumor types with the functional role of theseligands in cancer not known. A complete survey of human tumors for cellsurface calreticulin expression will be required to determine whetherthe regulation of the CD47-CRT phagocytic axis is a universal trait ofcancers.

One key question is raised by these studies: Why do cancers express cellsurface calreticulin, a pro-phagocytic signal? We have demonstrated thatcertain cancers evade the innate immune system by upregulatinganti-phagocytic signals, specifically CD47. One might expect cancers tosimultaneously downregulate pro-phagocytic signals to further increasetheir ability to evade macrophage phagocytosis. We propose two possibleexplanations. First, expression of cell surface calreticulin may be anunwanted consequence of cellular stress, whereby CD47 expression isupregulated to compensate and enable phagocytic evasion. In normalphysiology, cell surface calreticulin is induced on cells undergoing DNAdamage, marking these damaged cells for homeostatic phagocytosis. It ispossible that a small fraction of these cells may selectively avoidphagocytic clearance due to higher levels or upregulation of CD47, whichallows these damaged cells to survive and acquire additional mutations,eventually transforming into fully malignant cells. Several lines ofevidence support this. First, CD47 and CRT expression are highlycorrelated in several human tumors (FIG. 2A). Second, the smallpercentage of live cells that are calreticulin positive in some normalhuman tissue types (bone marrow and bladder) express higher CD47 levelsthan their calreticulin negative counterparts (FIG. 7). Third, thisincrease in CD47 expression appears to protect againstcalreticulin-mediated phagocytosis as knockdown of CD47 to 50% of wildtype levels enabled calreticulin-dependent phagocytosis (FIG. 2).

Expression of cell surface calreticulin may confer a pro-tumorigenicphenotype to cancer cells that is independent of phagocytosis. Thishypothesis is supported by the finding that increased calreticulinexpression in human tumors confers a worse clinical outcome acrossdisparate tumor types, tumor stage, and tumor-specific therapies (FIG.4). Cell surface calreticulin may allow more invasion and angiogenesis,as its ligand, LRP, is expressed on several vascular cell types. In tworeports, overexpression of calreticulin or calreticulin fragments intumor cell lines enhanced in vitro migration and invasion; however,other studies have reported alternative roles for calreticulin. In allof these studies the function of cell surface calreticulin was notdistinguished from its intracellular roles. Other possible tumorigenicroles include cell adhesion and immune escape through reduction of MHCclass I antigen presentation.

One key finding of our studies is the observation that increased levelsof calreticulin corresponds to a more aggressive tumor phenotype andconfers a worse clinical prognosis in several human malignancies. Giventhis finding and the restricted expression of cell surface calreticulinon tumor cells, calreticulin expression can be utilized in clinicaldiagnostics both in detection of cancer as well as monitoring ofresidual disease during therapy. Diagnostic modalities can include flowcytometry-based methods to detect cell surface calreticulin expressionin the blood or bone marrow for hematologic malignancies or throughimaging modalities utilizing a radio/fluorescent isotype coupled to cellsurface calreticulin for the localization of human solid tumors. Inaddition to diagnostic utility, cell surface calreticulin may serve as atherapeutic target for human cancers. First, therapeutic agents with anagonist function that activates calreticulin-LRP signaling can enablephagocytosis of tumor cells. This specifically includes the generationof an agonist calreticulin antibody. Second, calreticulin antibodies canbe coupled to a cytotoxic immunoconjugate to selectively target tumorcells given the tumor-restricted expression of calreticulin.

In summary, we have identified cell surface calreticulin as the dominantpro-phagocytic signal on several human cancers, which is absent on mostnormal cell counterparts and is required for anti-CD47 antibody-mediatedphagocytosis. These findings support the development of an anti-CD47antibody therapy for human malignancies, highlight the dynamicrelationship between pro- and anti-phagocytic signals in human cancer,and provide a rationale for the diagnostic and therapeutic roles oftargeting calreticulin.

Materials and Methods

Cell Lines and Human Samples.

MOLT4 and Daudi cell lines were obtained from the lab of Ronald Levy.639V was obtained from the DSMZ. All other cell lines were obtained fromthe American Type Culture Association (ATCC). Normal human bone marrowmononuclear cells were purchased from AllCells Inc. Normal peripheralblood and human cancer samples were obtained from patients at theStanford Medical Center with informed consent according to IRB-approvedprotocols: AML, ALL, and NHL human samples from Stanford IRB#76935,6453, and 13500, bladder cancer samples from Stanford IRB #1512,glioblastoma samples from Stanford IRB#9363, and ovarian cancer samplesfrom Stanford IRB #13939. Normal fetal bladder and brain cells werepurchased from ScienCell Research Laboratories.

Flow Cytometry Analysis.

For analysis of normal peripheral blood cells, normal bone marrow cells,AML, CML, ALL, bladder cancer, ovarian cancer, and brain cancer, thefollowing antibodies were used: CD34, CD38, CD90, CD45, CD31, CD3, CD4,CD7, CD11b, CD14, CD19, CD20, CD56, Glycophorin A (Invitrogen and BDBiosciences). Lineage negative (Lin−) was defined as CD3-CD19-CD20- forAML LSC and CD45-CD31- for GBM and bladder cancer CSC. Lin− was definedas CD3-CD4-CD7-CD8-CD11b-CD14-CD19-CD20-CD56-Glycophorin A- for NBM HSC,chronic phase CML GMP, CML CMP, and CML LSC. Analysis of CD47 expressionwas performed using an anti-human CD47 FITC antibody (clone B6H12.2, BDBiosciences). Analysis of human cell surface calreticulin expression wasperformed using mouse anti-human calreticulin conjugated to PE or FITC(clone FMC 75, Abcam). Human ERp57 expression was performed using apolyclonal rabbit anti-ERp57 antibody (Abcam) and then staining with adonkey anti-rabbit secondary antibody conjugated to PE (Ebioscience).

In Vitro Phagocytosis Assay.

Generation of human macrophages and in vitro phagocytosis assays wereperformed as previously described. Primary human samples or cell lineswere incubated with 10 μg/ml IgG1 isotype control (Ebiosciences), 10μg/ml anti-CD47 antibody (clone B6H12.2, ATCC), 4 μg/ml calreticulinblocking peptide (MBL International Coorporation), 10 μg/ml RAP(Fitzgerald Industries International), or 125 μg/ml recombinant CRThuman protein (Thermo Scientific). Per MBL, confirmation of blockingactivity was performed by Western blot analysis, verified by incubationof an anti-CRT antibody with 5 times higher concentration of the peptideand performing a Western blot analysis to determine if the specific bandhad been diminished. Cells were then analyzed by fluorescence microscopyto determine the phagocytic index (number of cells ingested per 100macrophages).

shRNA Knockdown of Raft Cells.

shRNA constructs targeting knockdown of human CD47 or a GAPD controlpackaged in the SMARTvector 2.0 lentiviral vector containing a turbo GFPreporter were purchased from Dharmacon, Inc. (Lafayette, Colo.). Viraltiters for each shRNA construct were greater than 10>8 TU/ml. Raji cellswere transduced with these lentiviral constructs, analyzed and sortedfor GFP expression, expanded, and sorted again for GFP expression forstable propagation of lentivirally-transduced cells. Knockdown of CD47protein levels was assessed by flow cytometry with anti-CD47 antibody(B6H12.2) with fold knockdown calculated by reduction in MFI normalizedover isotype control.

Xenotransplantation of Primary Human Cancer Cells into Mice.

For engraftment of human AML cells, AML LSC(CD34+CD38-CD90-Lin−) weresorted by fluorescence-activated cell sorting (FACS) and transplantedinto the facial vein of newborn NOD.Cg-PrkdcscidIl2rgtml/SzJ (NSG) mice,sublethally-irradiated with 200 rads. Leukemic engraftment was analyzed8 weeks later in the bone marrow of transplanted mice. For engraftmentof human bladder cancer, bulk bladder cancer cells were resuspended in25% matrigel (BD Biosciences) and transplanted subcutaneously into theflanks of adult NSG mice. Tumor volume was serially monitoredpost-transplantation by analyzing weights of excised tumors.

Analysis of Prognostic Value of Calreticulin in Human Malignancies.

Gene expression and clinical data were analyzed for six previouslydescribed cohorts of neuroblastoma, superficial and invasive urothelialcarcinoma of the bladder, and mantle cell lymphoma (see table 1 fordataset descriptions). Patients were stratified into high and lowcalreticulin expression groups based on the median expression levelwithin each cohort and analyzed for event-free, disease-specific, oroverall survival by Kaplan-Meier analysis. Subsequent dichotomous hazardratios, 95% confidence intervals, and log-rank p-values were analyzedreflecting estimates within Kaplan-Meier analyses (table 1).Additionally, analyses were performed based on continuous expression ofcalreticulin and clinical outcome as measured by log-likelihood p-valueswithin a univariate Cox regression model (table 1). Affymetrixmicroarray data were processed starting with CEL files, with Entrez Geneprobeset summarization using CustomCDF version 12 (49), andnormalization using MAS 5.0 linear scaling method. Overlapping samplesfrom related studies (FIGS. 4A,B and 4E,F), have not been removed.

What is claimed is:
 1. A method of targeting or depleting cancer cells,the method comprising: contacting cancer cells with an agent thatspecifically binds calreticulin in order to target or deplete the cells.2. The method of claim 1, wherein the cancer cells are contacted priorto treatment with a chemotherapeutic agent.
 3. The method of claim 1,wherein cancer stem cells are specifically targeted.
 4. The method ofclaim 1, wherein the cancer cells are leukemia cells.
 5. The methodclaim 4, wherein the cells are selected from AML, CML, ALL and NHL. 6.The method of claim 1, wherein the cancer cells are carcinoma cells. 7.The method of claim 6, wherein the carcinoma is a squamous cellcarcinoma or an ovarian carcinoma.
 8. The method of claim 1, wherein theagent is an antibody.
 9. The method of claim 8, wherein said antibody isconjugated to a cytotoxic agent.
 10. The method of claim 8, wherein theantibody is conjugated to a detectable label for imaging.
 11. The methodof claim 8, wherein the antibody is a bispecific antibody.
 12. Themethod of claim 11, wherein the bispecific antibody binds tocalreticulin and CD47.
 13. The method of claim 1, wherein the agent isan agonist of calreticulin-mediated phagocytosis.
 14. A method ofprotecting blood cells from phagocytosis, the method comprising:contacting said cells with a calreticulin blocking agent.
 15. The methodof claim 14, wherein the blocking agent is a CRT blocking peptide. 16.The method of claim 14, wherein the blocking agent is a CRT blockingoligosaccharide.
 17. The method of claim 14, wherein the blocking agentis a CRT blocking antibody.
 18. The method of claim 14, wherein saiddepleting is performed on said blood cells ex vivo.
 19. The method ofclaim 14, comprising: administering to an individual a compositioncomprising a population of hematopoietic cells and a calreticulinblocking agent that down-regulates phagocytosis.
 20. The method of claim19, wherein said cells are hematopoietic stem cells.
 21. The method ofclaim 19, wherein said cells are hematopoietic progenitor cells.
 22. Amethod of determining the fraction of normal cells susceptible toanti-CD47 based therapies through expression of calreticulin, so thatminimal potential toxicity to the patient will result.
 23. The method ofclaim 22, wherein said cells are hematopoietic stem and/or progenitorcells.
 24. The method of claim 22 wherein said cells are blood cellsand/or platelets.
 25. The method of claim 22 wherein said cells aretissue stem and/or progenitor cells detected in tissue biopsies,including intestinal crypt cells, lung cells.